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Standard Operating Procedures - North Campus

  1. Tissue Sample Preparation
  2. Cell Cycle Analysis
  3. Apoptosis Assays
  4. Proliferation Antigens
  5. Analysis of Oncogene Proteins
  6. Multidrug Resistance Assays
  7. Cytokine Receptors
  8. Other Flow Cytometric Assays
  9. Magnetic Cell Sorting
  10. Fluorescence in Situ Hybridization Techniques
  11. PCR Methods

1. Tissue Sample Preparation

1.1 Isolation of mononuclear cells from human peripheral blood or bone marrow aspirates

Reagents

  • Histopaque-1077 (Sigma # 1077-1)
  • Phosphate Buffered Saline (PBS)

Procedure

  1. To a 15-mL conical centrifuge tube, add 5.0 mL Histopaque-1077 and bring to room temperature.
  2. Prepare specimen mixtures in separate 15-mL tube: Mix 5 mL of blood/bone marrow aspirate with 5 mL of PBS.
  3. Delicately overlay blood/bone marrow over to Histopaque. Centrifuge at 400 x g for 20 minutes at room temperature with no brake.
  4. The mononuclear layer is visible as a white cloudy layer situated at the interface between the two solutions. Carefully aspirate the interface with a pasteur pipette and transfer to another 15-mL tube.
  5. Fill this tube with PBS and centrifuge at 500 x g for 5 minutes.
  6. Aspirate the supernatant and discard.
  7. Resuspend cell pellet with 5.0 mL PBS and repeat steps 5 and 6.
  8. Discard supernatant and resuspend cell pellet in 1 mL of PBS.

1.2 Neutrophil isolation from human peripheral blood

Reagents

  • Histopaque 1119 (Sigma #1119-1)
  • Histopaque 1077 (Sigma 1077-1)
  • Phosphate Buffered Saline Solution (PBS)
  1. Add 3 mL Histopaque 1119 to a 15-mL conical centrifuge tube.
  2. Carefully layer 3 mL of Histopaque 1077 onto the Histopaque 1119.
  3. Carefully layer 6 mL of whole blood onto the upper gradient of the tube from step 2.
  4. Centrifuge at 700 x g for 30 minutes at room temperature.
  5. Carefully remove centrifuge tubes. Two distinct opaque layers should be observed.
  6. Aspirate and discard fluid to within 0.5 cm of layer A. Transfer cells from this layer to a tube marked "mononuclear cells".
  7. Aspirate and discard remaining fluid to within 0.5 cm of layer B. Transfer cells from this layer to a tube labeled "granulocytes".
  8. Wash the cells by addition of 10 mL PBS. Centrifuge 10 minutes at 500 x g. Remove the supernatant and discard.
  9. Resuspend the cells by gentle aspiration with a Pasteur pipette.
  10. Repeat steps 8 and 9.
  11. Resuspend cells in an appropriate volume of PBS and use as desired.

1.3 Isolation of total white cells from human peripheral blood by red cell lysis

Non-fixing lysing solution

Ammonium Chloride Lyse

10X concentration

Dilute 10 mL stock with 90 mL double distilled water. Store at 4°C.

  • NH4Cl (ammonium chloride) 80.2 g
  • NaHCO3 (sodium bicarbonate) 8.4 g
  • EDTA (disodium) 3.7 g

Working solution

QS to 1 liter with double distilled water. Store stock at 4°C for up to six months.

Procedure

  1. Obtain a whole specimen in a 10-mL heparinized tube, preferably preservative free.
  2. Aliquot 2 mL into each of four 50-mL conical centrifuge tubes.
  3. Immediately fill tubes up to 50 mL with cold 1X lysing solution.
  4. Rock for 10 minutes at room temperature.
  5. Spin down at 4°C for 10 minutes at 250 x g.
  6. Decant supernatant and allow tubes to drain briefly.
  7. Resuspend pellet by drawing tube across tube rack (i.e., scrape the pellet).
  8. Combine two pellets into one 50-mL tube. Fill to 10 mL with cold PBS to wash.
  9. Spin, decant and scrape pellet as above.
  10. Combine to the two pellets into one tube and wash with 10 mL cold PBS.
  11. Spin, decant and scrape pellet. Use cells as needed.

Note: This osmotic lysis method does not harm the white cells or alter membrane asymmetry. Due to the osmotic method of lysis, the 10X stock must be diluted to 1X with water. Using a buffer such as PBS alters the osmolarity of the lysis buffer and it will no longer be effective. This lysis technique will leave all populations of white cells, including granulocytes.

Contributed by: Virginia Snell

Further Reading: Modified from the Handbook of Flow Cytometry Methods, J. Paul Robinson, editor, Wiley-Liss Inc.

1.4 Preparation of single cells from solid tissues

A common feature of all flow cytometric studies of solid tissues is the requirement from dispersal into single cells (or nuclei) before staining and analysis. Dispersal of solid tumors for flow cytometric analysis can be achieved by a variety of methods, most of which employ a combination of mechanical and enzymatic disaggregation. The choice of dispersal method depends on the type of material to be examined and the point to be measured. Also, special treatment is required for:

1.5 Cell Counts (Hemacytometer)

Hemacytometer grid system

Errors can be introduced in a number of ways: dilution errors, loss of cells during pipetting, uneven suspension of cells, overfilling or underfilling of the chamber and the counting of cells before they settle. Random distribution of cells in the chamber is another source of error but can be compensated for by counting a large number of cells. Clean chamber just before and just after use. If the chamber is loaded too heavily, clean it and begin again; do not attempt to remove excess liquid.

Example: Sample of cell suspension diluted 1:1 with trypan blue

Grid #1 (16 squares)= 195 cells
Grid #2 (16 squares)= 177 cells
Grid #3 (16 squares)= 204 cells
Grid #4 (16 squares)= 166 cells
total = 742 cells
mean = 185.5 cells

Formula:
Cell concentration (cells/mL)= mean x (10) x dilution factor
= 185.5 x (104) x 2
= 3.71 x 106 cells/mL

1.6 Thawing Frozen Cells

  1. Quickly thaw frozen vial of cells by brief incubation of tube at 37°C in a water bath until sample is just slushy. Wipe tube well with 70% ethanol.
  2. Transfer sample to 15-mL conical tube containing 10-13 mL RPMI 1640 complete medium warmed to 37°C.
  3. Centrifuge cells immediately at 800 x g. Remove supernatant.
  4. Wash cells 2X with 10 mL RPMI complete. Centrifuge cells.
  5. Resuspend cells in desired buffer or medium and use as needed.

Notes: DMSO is a polar planar molecule which is not only a differentiating agent but is also toxic to the cells. This toxicity is marked at warm temperatures. It is very important, therefore, to remove the cells from the freezing medium as rapidly as possible. This is accomplished by diluting the thawed cell suspension immediately into a large volume of medium and centrifuging the cells to remove the diluted DMSO.

1.7 Freezing Samples and Cell Lines

  1. Either lyse RBCs or Ficoll cells using sterile methods. Wash cells 1X with cold sterile medium.
  2. Resuspend cells in an amount of cold medium. Count cells. Centrifuge sample.
  3. Cells should be frozen in aliquots of 10-20 x 106 cells. Resuspend pellet in 500 µL of cold medium per tube.
  4. Label cryotubes with appropriate information:

    Cell Lines:

    Name of cell line
    Date
    Passage number (if known)
    Source of cells
    Number of cells

    Primary tissue:

    Last name and first initial of patient
    MD Anderson number
    Date
    Diagnosis
    Tissue type (BM, PB, Ph)
    Treatment (F= ficolled, L= lysed)

  5. Add an equal amount of freezing medium (recipe following) to the RPMI drop-wise while gently swirling. The ratio of RPMI cell suspension to freezing medium should always be 1:1.
  6. Add 1 mL of the resulting cell suspension to the appropriate crytubes. Put tubes on ice.

For freezing in the -70°C freezer

  1. Place tubes in a cryobox which has been cooled to 4°C.
  2. Place cryobox into -70°C freezer.
  3. Remove frozen vials the next day to liquid nitrogen freezer.
  4. Store empty cryobox in refrigerator.
  5. For freezing in controlled freezer:
    • You may need a pilot tube as an internal control for the degreed freezer. In a cryovial place 500 µL RPMI and 500 µL freezing medium. Put on ice with prepared tubes.
    • Cool the degreed freezer chamber to 4°C.
    • Place cryovials into chamber. The freezing process takes about an hour.
    • Remove tubes immediately to liquid nitrogen freezer.

Freezing Medium

40 mL Hank's Buffered Saline Solution (HBSS) - Ca+2, -Mg+2
20 mL DMSO
40 mL fetal calf serum
100 mL

Notes: DMSO is a polar planar molecule which is not only a differentiating agent but is also toxic to the cells. This toxicity is marked at warm temperatures. It is very important therefore, to add the DMSO as the last step, keep cells on ice once DMSO is added, and start the freezing process as soon as possible. DMSO keeps cells viable during freezing by preventing the water within cells from crystallizing and bursting the lipid membrane.

2. Cell Cycle Analysis

2.1 Surface Antigen/DNA (Counterstaining with PI)

Assessing the cell cycle expression of a surface marker by propidium iodide starts with the standard procedure for staining the desired sample with the desired antibody and its isotype control. The cells must then be fixed prior to counterstaining, and two procedures are available: ethanol- or paraformaldehyde-based fixations. The method used should depend on the importance of the phenomena to be measured. Ethanol fixation can offer the investigator an excellent CV for DNA analysis while the paraformaldehyde technique followed by ethanol is better for preserving the surface marker fluorescence.

  1. The ethanol based fixation:

    Add 0.7 mL of ice cold 100% ethanol to 1 x 106 stained cells contained in 0.3 mL cold PBS. Incubate at -20°C for 30 minutes or overnight. Centrifuge and remove supernatant.

    -or-

    The paraformaldehyde fixation:

    Add 0.5 mL of 2% paraformaldehyde in PBS to 1 x 106 stained cells in 0.5 mL PBS. Incubate at 4°C for 30 minutes. Centrifuge and wash once with cold PBS. Samples can be counterstained immediately or stored at 4°C for up to one month.

  2. Add 250 µL of 500 units/mL Rnase in PBS with 1.12% sodium citrate to cell pellet. Vortex gently and incubate at 37°C for 30 minutes.
  3. Add 250 µL of propidium iodide (Sigma) at 50 mg/mL to sample. Incubate at room temperature for at least 1 hour in the dark.
  4. Read samples on flow cytometer.

2.2 Two surface antigens/DNA (counterstaining with DAPI)

  1. 2 x 106 cells are stained with two monoclonal antibodies labeled either FITC, PE or PERCP. Follow standard procedure for staining with surface markers. Process appropriate isotype controls in parallel.
  2. Fix cells for 30 minutes with 0.5% paraformaldehyde (1 mL) at 4°C.
  3. Centrifuge cells at 500 x g for 5 minutes.
  4. Resuspend cells in 0.1% Triton-X-100 (in PBS) for 3 minutes at room temperature.
  5. Centrifuge cells and wash twice with PBS.
  6. Add DAPI to achieve a final concentration of 1 µM; incubate for 1 hour or overnight in the dark.

2.3 DNA-RNA Differential Staining Using Acridine Orange

Reagents

  • Stock solution AO: 1 mg/mL
  • Phosphate-citric acid buffer (pH 6.0)
    • 37 mL 0.1 M Citric Acid
    • 63 mL 0.2 M Na2HPO4
  • Solution A
    • 0.1 mL Triton X-100 (0.1% final)
    • 8.0 mL 1N HCl (0.08 N final)
    • 0.877 g NaCl (0.15 N final)
    • QS to 100 mL with dH2O. Store for 6 months at 4°C, in dark.
  • Solution B
    • 100 mL Phosphate-Citric Acid Buffer
    • 0.877 g NaCl (0.15M final)
    • 34 mg Disodium EDTA (1mM final)
    • 0.6 mL Stock AO (20 µM)
    • Store for 6 months at 4°C in dark.
    • For dispensing bottles use 0.4 mL for Solution A and 1.2 mL for Solution B (in foil wrapped bottles)
  • Nuclear isolation medium:
    • 10 mM Tris Buffer (pH 7.6)
    • 1 mM Sodium Citrate
    • 2 mM MgCl2
    • 0.1% (v/v) Nonionic Detergent Nonident P-40

Staining unfixed cells

  1. Transfer 200 µL cell suspension (at most 2 x 105 cells) to FACS tube.
  2. Chill on ice. Add 400 µL Solution A gently and wait 15 seconds on ice.
  3. Add 1.2 mL of ice-cold Solution B gently and measure cell luminescence over next 3-15 minutes.

Fixed cells

  1. Fix cells in 70% ethanol at 4°C.
  2. Wash cells (250 x g, 10 minutes) twice to remove ethanol and resuspend in PBS at 2 x 106/mL.
  3. Stain 200 µL cell suspension using Solution A and B as described above. (Triton X-100 is not necessary with fixed cells).

To estimate the specificity of the reaction, remove ethanol and resuspend cells in PBS. Incubate with RNAse (103 U/mL) for 30 min at 37°C prior to AO staining. Compare with the sample not treated with RNAse.

Note 1: AO concentration is critical. If cell number is high (>2 x 106/mL), the amount of bound AO is proportionally high and free dye may be reduced. RNA denaturation may be incomplete and some RNA stains green. Therefore, dilute cell suspension.

Note 2: If measurements take place outside the nozzle (i.e., in air), dye diffusion occurs after the stream leaves the nozzle. This can lower the actual AO concentration. Therefore, increase AO concentration up to 60 µM in Solution B and increase flow rates. It is important to determine correct concentration and flow rate for individual machines.

Note 3: 

Filters: Use 640- or 650-nm long pass filters rather than 610 or 620 nm for AO bound to RNA (reduces DNA component).

Note 4: Most commercial instruments work well. The following instruments require higher AO concentration (i.e., about 25-60 µM) to compensate for dye diffusion: FACS II, FACS II, FACStar, EPICS IV and C and 30-H Cytofluorograf.

Note 5: For the Ortho-50 H, the non-sorting channel is better than the sorting one.

Acquisition and Analysis

When acquiring a sample for acridine orange, The doublet discrimination (DDM) function of the FACScan must be on. The DDM settings will be for FL1, as AO fluoresces green when bound to DNA. The gain settings for AO are ideal when set at 1. It is very seldom that these gains need to be adjusted. The parameters that are acquired are FL1 Area, FL1 Width, FL3 Height in the linear mode and FL1 Height in log mode. Five thousand cells are usually sufficient for this measurement.

When setting the machine with a sample, it is best to use a control cell population. Although the scatter properties of the cells are usually not of interest after permeabilization, the first parameters adjusted should be the SSC v. FSC to ensure that all cells are being acquired, and to eliminate unnecessary acquisition of debris. The second setting adjusted should be the FL1 Area v. FL1 Width. It is traditional that the G0-G1 peak be set at about 200 in FL1 Area. Next, looking at either FL1 Height v. FL3 Height or FL1 Area v. FL3 Height, set the RNA (FL3) so that the G0 RNA level is closer to the left and the G2M RNA does not extend into the right side, basically in the center of the screen. At this point, the sample may be acquired, and should need no or little adjustment for other samples. If using multiple cell lines, the different lines may need to be adjusted separately. For 'Analysis See', show the FL1 Area v. FL1 Width. Make a gate around the singlets. Show FL1 Height v. FL3 Height, with logical gate on G1. Make another gate to exclude the dead cells in the lower left of the hypodiploid area of FL1 Height. On 'Gate Specs', change G3=R3 to G3= R1 AND R2. Show a histogram of FL1 Height, with logical gating on G3. You must combine the logical gates as R1 and R2 or the gates will not be combined. Make a marker on the hypodiploid region of the histogram. The resulting percentage is the percent apoptotic cells.

Contributed by: Virginia Snell

Further Reading:

Darzynkiewicz, Z. Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. In Methods in Cell Biology, Vol 33. Z. Darzynkiewicz & H. Crissman, eds. Academic Press, New York, 1990. pp 285-298.

Taken from the Handbook of Flow Cytometry Methods, J. Paul Robinson, editor, Wiley-Liss Inc.

2.4 Chromatin denaturation

This technique which aims at the detection of M-phase cells is based upon the fact that cells in different cell cycle phases are not equally sensitive to partial acid denaturation.

  1. Fix 10e6 cells in 500 µL PBS by adding 4.5 mL cold 70% ethanol in a glass tube. At this point, samples can be stored at 4°C for future use.
  2. Centrifuge cells, remove supernatant and resuspend in 1 mL HBSS.
  3. Treat with 100 µL highly purified RNAse (for example, Worthington) diluted 1:5000. Incubate at 37°C for 45 minutes.
  4. Mix 200-µL aliquots with 500 µL buffer containing 0.1M KCl and 0.1M HCl at pH 1.4.
  5. Add 2.0 mL of 5 µg/mL acridine orange in a buffer containing 0.1M citric acid, 0.2M Na2HPO4 at pH 2.6.
  6. Measure immediately on flow cytometer.

2.5 Staining nuclei with BrdUr/IdUr and propidium iodide (PI): nuclear isolation method

  1. Label cells with IdUr or BrdUr at a concentration of 10 µg/mL for 30 min at 37°C. Wash 1X with PBS at 1500 rpm for 5 minutes.
  2. Resuspend pellet in 70% ethanol while gently vortexing to fix cells. Store cells at 4°C for at least 24 hours or up to one month.
  3. Isolate cell nuclei:

    Incubate cells in 5 mL 0.025-0.01% pepsin (Sigma) w/v in 0.01N HCl for 1 hour or optimal time at 37°C. .

    Vortex cell suspension vigorously for 30 seconds for single cell suspensions. For solid tumors, cell suspension may need to be passed through a 18-gauge needle and filtered through 35-µm nylon mesh.

    Centrifuge at 2000 rpm for 5 minutes (Sorvall Technospin).

  4. Resuspend pellet in 1 mL of 2N HCl for 20 minutes at 37°C.
  5. Neutralize HCl with 2 mL of 0.1 M sodium tetraborate (use 2X the volume of 2N HCl). Centrifuge at 2000 rpm for 5 minutes. Wash 2X in PBTB (2000 rpm/5 minutes).
  6. Staining sample for BrdUr:

    Resuspend 2e6 nuclei in 150 µL BrdUr specific antibody (Caltag) diluted 1:500 v/v in PBTB for 1 hour at room temperature in the dark. [We are currently using BR3 from another source (Dolbeare) diluted at a concentration of 1:10, to be used at a final concentration of 1:500, so use the diluted stock 1:50].

    Wash 2X with PBTB (2000 rpm/5 minutes).

    Resuspend pellet in 150 µL GAM-FITC IgG (Sigma) diluted 1:50 v/v in PBTB and incubate for 1 hour at room temperature in the dark.

    Wash 2X with PBTB (2000 rpm/5 minutes).

  7. Staining Sample FOR IdUr:

    Resuspend 2e6 nuclei in 20 µL of IdUr/BrdUr specific B44-FITC antibody (Becton Dickinson) diluted 1:2 v/v in PBTB and incubate for 1 hour at room temperature in the dark.

    Wash 2X with PBTB (2000 rpm/5 minutes).

  8. Resuspend nuclei in 10 µg/mL PI in 2 mL PBTB. Store nuclear suspension at 4°C for at least one hour, preferably overnight.
  9. Add Rnase 30 minutes (37°C) prior to flow at a final concentration of 10 µg/mL.

Notes
Obviously, since pepsin is used in this procedure to strip the cytoplasm from the cell nuclei, it is necessary to have as homogeneous a cell population as possible as cell identity will be lost. There are other methods which do not use pepsin digestion, but this method gives the best CV on PI staining and the most separation between labelled and unlabelled IdUr and BrdUr. These conditions are ideal when analysis of cell cycle kinetics are desired, such as the potential doubling time (Tdpot) and duration of S phase (Ds). If one only desires the labelling index (percentage of cells labeled with nucleic analog) and/or cell surface staining, another method which does not use pepsin digestion is more appropriate.

Almost all methods of staining with BrdUr or other nucleic analogs use slight variations of the same techniques. In all methods, the first step is to label the cells with the analog. The method of incubation, i.e., using pulse or pulse/chase or multiple labelling is discussed later. Second, this method uses pepsin digestion to strip the cytoplasm from the nuclei. Pepsin is naturally found in the stomach, in the presence of dilute HCl. Pepsin must have dilute HCl and must be incubated at 37°C to work most efficiently. Pepsin is a proteolytic enzyme, so it basically punches holes of the phospholipid membrane by eating the transmembrane proteins. This is why it is important to vortex the cells after pepsin incubation -- to shear the remaining phospholipid membrane and cytoplasm from the nuclei. The most difficult part of this method is optimizing the pepsin digestion. This must be done for every cell type. Delicate single cells such as lymphocytes need lower pepsin concentrations and shorter incubation times than cells from solid tumors. Underdigestion will not allow stripping of all cytoplasm. Overdigestion will begin to degrade the viability of the nuclear membrane. An optimization procedure follows later. The best way to test optimization is to view the cells under a fluorescent microscope after staining with PI to observe the state of the nuclei. The third step, one common in all methods, is incubation with relatively concentrated (2N) HCl. This step is very important, as it dissociates the DNA and makes the incorporated analog accessible to the antibody and relatively large fluorochrome. The use of a buffer with a detergent such as Tween-20 or Triton-X keeps everything accessible.

Contributed by: Virginia Snell

References:

White, R.A., A. Pollack, and N.H.A Terry (1994) Simultaneous Cytokine Measurement of Aneuploid Tumors and Associated Diploid Cells Following Continuous Labelling with Chlorodeoxyuridine. Cytometry 15:311-319.

White, R.A. and N.H.A Terry (1992) A Quantitative Method for Evaluating Bivariate Flow Cytometric Data Obtained Using Monoclonal Antibodies to Bromodeoxyuridine. Cytometry 13:490-495.

Terry, N.H.A, R.A. White, M.L. Meistrich, and D.P. Calkins (1991) Evaluation of Flow Cytometric Methods for Determining Population Potential Doubling Times Using Cultured Cells. Cytometry 12:234-241.

Terry, N.H.A., R.A. White, and M.L. Meistrich () Cell Kinetics: from Titriated Thymidine to Flow Cytometry BJR Suppl 24:153-157.

Terry, N.H.A. (1995) The Predictive Value of Tumor-Cell Kinetic Parameters in Radiotherapy: Considerations Regarding Data Production and Analysis. Journal of Clinical Oncology 13:1833-1836.

J.C. Carlton, N.H.A. Terry and R.A. White (1991) Measuring Potential Doubling Times of Murine Tumors Using Flow Cytometry. Cytometry 12: 645-650.

Pollack, N.H.A. Terry, C.S. Wu, B.M. Wise, R.A. White and M.L. Meistrich (1995) Specific Staining of Iododeoxyuridine in Tumors Double Labelled in Vivo: A Cell Kinetic Analysis. Cytometry 20: 53-61.

2.6 Cell cycle kinetics of BrdUTP incorporated cells using UV photolysis in whole cells: an alternative method of using BrdUTP without DNA denaturation

  1. Cells are labeled with BrdUTP (Sigma, St Louis, MO, USA) at a concentration of 10 µM for 60 minutes (Time may be varied according to cell type and culture conditions.).
  2. Cells are washed once with cold HBSS.
  3. The cell pellet is resuspended in 2 mL of cold HBSS and the cell suspension is transferred to 60 x 15-mm polystyrene Petri dishes (Corning NY, USA). The dishes are then placed directly on the glass surface of a Fotodyne UV 300 analytic DNA transilluminator, containing four 15-W bulbs (Fotodyne, Inc., New Berlin, WI, USA) providing maximal illumination at 300-nm wavelength. Cells are exposed to UV light for 5 minutes.
  4. Cells are then centrifuged, fixed in 70% ice-cold methanol and stored at 4°C for up to four days.
  5. Cells are rehydrated by washing twice in PBS.
  6. The cell pellet (about 1 x 106) is blotted to remove all extraneous supernatant as possible. The pellet is resuspended in 50 µL of TdT reaction buffer containing: 10 µL of 5X buffer solution (1 M potassium cacodylate; 125 mM Tris-HCl, pH 6.6; 1.26 mg/mL bovine serum albumin (BSA); 5 µL of 25 mM cobalt chloride; 0.5 µL (12.5 units) of TdT (all from Boehringer Mannheim, Indianapolis, IN, USA), and 0.25 nmoles of BrdUTP (Sigma) is added.
  7. Cells are incubated at 37°C for 60 minutes. The reaction is stopped by washing the cells twice in 15 mM EDTA-NaOH, pH 7.8.
  8. The cells are then incubated in 100 µL of a solution containing 0.7 µg of FITC-conjugated anti-BrdUrd monoclonal antibody (Becton Dickinson, San Jose, CA, USA, clone B44), 0.1% Triton-X-100 (Sigma) and 1% BSA. Cells are washed twice with the above buffer (minus the monoclonal antibody).
  9. Cells are counterstained with 5 µg/mL PI in the presence of RNase as described (Li et al., 1995, Cytometry 20:172).

References: Li, X, Troganos, F., Melamed, M.R., and Darzynkiewicz, Z. (1994) Detection of 5-bromo-2-deoxyuridine incorporated into DNA by labeling strand breaks induced by photolysis (SBIP). Int. J. Oncol. 4:1157-1161.

2.7 Bromodeoxyuridine (BrdU)/DNA: whole cell method

  1. Culture conditions and equipment:
    • 250 mL culture flask wrapped in aluminum foil
    • 25 mL Iscoves Medium
    • 10 mM BrdUr
    • Culture 1e7 Ficoll-separated cells for 30 minutes at 37°C
  2. Centrifuge at 200 x g for 10 minutes at 10°C. Remove supernatant.
  3. Fix pellet with 2 mL ice cold 70% ethanol. Store at 4°C for up to one month.
  4. Centrifuge at 200 x g for 10 minutes at 10°C. Remove supernatant.
  5. Add 2 mL ethanol pH_ TB for 30 minutes at 4°C.
  6. Add 2 mL PBTB.
  7. Centrifuge at 200 x g for 10 minutes at 10°C. Remove supernatant.
  8. Add 2 mL PBS (pH 7.2).
  9. Centrifuge at 200 x g for 10 minutes at 10°C. Remove supernatant.
  10. Add 250 mL PBS (pH 7.2) + 20 mg/mL propidium iodide.
  11. Measure cells on FACScan for BrdUr/DNA content.

Contributed by: Dr. Hans-Dieter Kleine

2.8 Surface marker staining combined with Hoechst 33342

  1. Ficoll bone marrow according to previous protocol.
  2. Adjust the cell concentration to 5 x 104 cells/µL in PBS.
  3. Use 50 µL of this cell suspension for the following steps or multiply all mentioned amounts with an appropriate number.
  4. Incubate 30 minutes on ice with antibodies to surface antigens: isotype controls are prepared the same way as for normal immunophenotyping.
  5. Wash twice with 2 mL of PBS.
  6. Resuspend the pellet in 1 mL PBS, vortex gently.
  7. Add 1 mL of 2% ice cold paraformaldehyde in PBS while vortexing the sample (final concentration 1%). Incubate on ice for 1 hour. Wash twice with PBS.
  8. Resuspend the pellet in 1.9 mL of 0.9% normal saline solution. Incubate on ice for 30 minutes then add 0.1 mL Tween-20 10% in normal saline, vortex gently.
  9. Add 60 µL of Hoechst 33343 stock solution (100 µg/mL in normal saline). Incubate overnight in the refrigerator.
  10. Read samples on flow in the morning.

The CV of the G0/G1 peak should be less than 5%, provided the fluorochrome spheres yielded a CV less than 4% on the UV parameter. Scatter and staining properties should not differ significantly from fresh stained samples.

2.9 Modified Hoechst method for bcl-2 staining

Repeat steps 1 - 7 above.

 8. Resuspend the pellet in 1.9 mL of PBS. Add 0.1 mL of 10 % Tween-20 in normal saline, vortex gently. Incubate on ice for 15 minutes.

 9. Wash twice in PBS.

10. Add 10 uL of bcl-2 FITC ( DAKO ) antibody in 50-90 µL of cell suspension, and incubate on ice for 15 minutes. Also add 10 µL of isotype control to the appropriate tube.

11. Wash twice in normal saline and resuspend in 1.9 mL of normal saline.

12. Incubate for 30 minutes on ice.

13. Add 0.1 mL Tween-20 10% in normal saline, vortex gently. Add 60 µL of Hoechst 33343 stock solution (100 µg/mL in normal saline). Incubate overnight in the refrigerator, and run the sample in the morning.

2.10 Hoechst staining procedure for detection of CD34- side population cells

  1. Lyse whole bone marrow with non-fixing lysing solution. Rock gently for 5-10 minutes.
  2. Spin down (1500 rpm/5 minutes). Note: You may need to add lysing solution again for 1 minute if there are red cells in the pellet.
  3. Wash cells with warm medium (RPMI 1640 +1 mM HEPES, + 2% FCS + pen/strep).
  4. Count the number of cells.
  5. Make a cell suspension in medium of 2.5 x 106 cells/mL.
  6. Aliquot 1 mL of this cell suspension into the following tubes:

    #1) IgG1-FITC/ IgG1-PE/ IgG1-TC

    #2) IgG1-FITC/ IgG1-PE/ IgG1+ IgG2a-GAM-TC

    #3) IgG1-FITC/ IgG1-PE/ LINEAGE-GAM-TC

    #4) CD45-FITC/ IgG1-PE/ IgG1+ IgG2a-GAM-TC

    #5) IgG1-FITC/ CD38-PE/ IgG1+ IgG2a-GAM-TC

    Place these tubes on ice to stain for compensation.

  7. Aliquot 4 mL of cell suspension into the following tubes:

    X4 #6 (A,B,C,D) CD34-FITC/ CD38-PE/ LINEAGE-GAM-TC

    X3 #7 (A,B,C) CD34-FITC/ CD95-PE/ LINEAGE-GAM-TC

    X1 #8) (IgG1/4E3)/ CD34-PE/ LINEAGE-GAM-TC

    X1 #9) (IgG1/4E3)/ CD34-PE/ CD38-TC

    Add the Hoechst dye at 20 µL per 4 mL. Incubate at 37°C in the dark for 90 minutes.

  8. Spin the cells down, from step 5 and 6. Wash 1X with cold buffer (HBSS + 1 mM HEPES + 2% FCS + pen/strep).
  9. Resuspend the pellet in 100 µL of cold washing solution. Place tubes #1 and 9 on ice. Add 51 µL of the lineage cocktail to the appropriate tubes:

    The lineage cocktail is made up of the following antibodies:

    CD61 Caltag 1 uL per test CD56 Caltag 5 µL

    CD16 Caltag 1 uL CD33 BD 5 µL

    CD10 Caltag 1 uL CD66b Immunotech 5 µL

    CD19 Caltag 2 uL CD4 Immunotech 10 µL

    CD11b Caltag 2 uL CD8 Immunotech 5 µL

    CD41 Caltag 2 uL CD22 Immunotech 5 µL

    CD13 Caltag 2 uL GlyA Dako 5 µL

    For control tubes, add 3 µL each of pure IgG1 and IgG2a from Caltag.

  10. Incubate cells on ice for 15-30 minutes. Wash 1X with 2-3 mL cold buffer. Resuspend the pellet in 100 µL cold buffer. Add 10 µL GAM-TC per tube. Incubate on ice for 15-30 minutes. Add 2 µL of GAM-TC to the compensation tubes. Wash 1X with 2-3 mL cold buffer.
  11. Resuspend pellet in 80 µL cold buffer. Add 20 µL mouse serum. Incubate 15 minutes on ice. The mouse serum blocks any free receptor sites on the GAM, and is necessary when staining with other mouse anti- human antibodies after the GAM. Wash cells 1X with 2-3 mL cold buffer.
  12. Resuspend pellet in 100 µL. Add 100 µL additional buffer to tubes #8 and 9. Split these tubes into two additional tubes (A and B). Add 5 µL IgG1-FITC to tubes A and 5 µL 4E3-FITC (Signet) to tubes B. Add the appropriate other directly conjugated antibodies. Incubate 15-30 minutes, wash 1X with 2-3 mL cold buffer.

    Split tubes:

    #8A) IgG1-FITC/ CD34-PE/ LINEAGE-GAM-TC

    #8B) 4E3-FITC/ CD34-PE/ LINEAGE-GAM-TC

    #9A) IgG1-FITC/CD34-PE/ CD38-TC

    #9B) 4E3-FITC/ CD34-PE/ CD38-TC

  13. Resuspend pellet in 4 mL cold buffer. Store at 4°C until sorting.

3. Apoptosis Assays

3.1 Acridine orange staining for apoptosis

Follow the same protocol used for cell cycle analysis of cells. The apoptotic population of cells will have the same RNA content of live cells, but will have a sub-G0/G1 DNA content.

3.2 Propidum iodide staining for apoptosis

Follow the same used for cell cycle analysis of cells. The apoptotic population of cells will have a sub-G0/G1 DNA content on the DNA histogram.

3.3 Detection of phosphotidyl serine (PS) using Annexin V-FITC

APOPTEST-FITC apoptosis kit (Nexins Research B.V, #A700)

This protocol is for the simultaneous detection of surface markers and phosphatidyl serine exposure by binding of Annexin V-FITC.

  1. This step is for patient samples only, not needed for cultured cells. Lyse peripheral blood or bone marrow with nonfixing lysing solution according to protocol.
  2. This step is for patient samples only, not needed for cultured cells. Incubate cells in 15 mL 0.9% normal saline containing 5 mM EDTA (pH 7.4) for 15 minutes on ice. This releases any endogenous Annexin V which is bound to the PS, as well as releasing bound platelets which contribute to false positivity. Wash cells once with cold PBS.
  3. Aliquot cells into appropriately labeled tubes to stain for PE and/or PERCP or TRI conjugated surface markers according to appropriate protocols.
  4. Wash cells once with cold PBS. Blot pellet very well removing supernatant as not to disrupt the calcium concentration of the binding buffer to be added.
  5. Add 200 µL of binding buffer to pellet (about 105-106 total cells). Add 2 µL Annexin V-FITC and incubate on ice for 10 minutes in the dark. Retain tubes with no Annexin and use autofluorescence of cells as negative control for flow. Read samples directly.
  6. Optional: Add propidium iodide (stock: 100 µg/mL) to the Annexin/binding buffer solution at a final concentration of 5 µg/mL. Incubate 10 minutes on ice and read flow directly.

Special Considerations:
Only fresh samples are used for spontaneous apoptosis, no overnight storage or use of frozen cells. Do not fix or permeabilize cells, as this will induce false positivity due to exposure of PS on the inner leaflet of the cell membrane. The use of binding buffer during staining of Annexin V is crucial because Annexin requires a physiological concentration of calcium to bind. Alternatively, cell culture medium or HEPES buffer may be used, as they contain the same calcium levels as the binding buffer. Do not use binding buffer older than four weeks as it may cause false positive results, possibly due to pH problems. It is desirable to use propidium iodide to distinguish necrotic cells (false positive) from apoptotic cells.

Contributed by: Dr Oliver Kliche/Virginia Snell

References:

Fadok VA, Voelker DR, Campbell PAS, Cohen JJ, Bratton DL and Henson PM (1992) Exposure of phosphotidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148, 2207-2216.

3.4 Detection of apoptotic cells using TUNEL for flow, with surface staining and/or DNA staining optional

  1. Optional: For surface marker staining, stain cells with desired surface markers and appropriate isotypic control for 30 minutes on ice. Wash one time with PBS.
  2. Fixation: Mix one part 2X PBS with one part 2% paraformaldehyde (methanol-free). Add resulting 1% paraformaldehyde slowly to cells while gently vortexing. Incubate at 4°C for at least one hour. If using PE-Cy5 tandem conjugate dyes as surface markers, best result are achieved if cells remain in 1% paraformaldehyde until TUNEL procedure.
  3. Wash 2 times with 4 mL PBS.
  4. Permeabilization: If surface marker staining is not done, fix cells with ice cold 70% ethanol while gently vortexing. Cell may be stored at -20°C in 70% ethanol for two weeks or longer. If surface markers are used, best results are achieved by permeabilizing the cell membrane with Triton-X. Resuspend the pellet 500 µL of 0.1% Triton-X in PBS, and vortex gently. Incubate on ice for 15 minutes. Wash two times with 4 mL cold PBS.
  5. Count the number of samples. Make enough buffer for at least one more than the number of actual samples. Add appropriate amount of biotin-16-dUTP and TdT enzyme to buffer (recipe for all follows). Add 40 µL of the reaction buffer to cells. The control reaction is buffer plus biotin-16-dUTP without the TdT enzyme. Incubate tubes at 37°C for one hour. Wash cells twice with 4 mL of PBS.
  6. Make enough avidin mix for at least one more sample than the actual number of samples (recipe follows). Add 100 µL of avidin mix to samples. Incubate at room temperature in the dark for 30 minutes. Wash twice with 4 mL of PBS.
  7. Resuspend cells in PBS for flow cytometry.
  8. Optional: for cell cycle analysis, add propidium iodide to a final concentration of 5 µg/mL. Incubate at room temperature for 30 minutes in the dark. Read on flow cytometer.

    TUNEL Buffer

    • 20 µL 5X buffer
    • 10 µL 10X CoCl
    • 70 µL dH2O

    TUNEL Buffer Reaction Mixes

    TdT Reaction Mix

    • 40 µL TUNEL buffer
    • 0.6 µL biotin-16-dUTP
    • 0.3 µL TdT enzyme per individual sample

    Control Reaction Mix

    Make fresh.

    • 40 µL TUNEL buffer
    • 0.6 µL biotin-16-dUTP
    • 100 µL buffer per individual sample

    Avidin

    Avidin Buffer

    Filter and store at 4°C.

    • 4X SSC
    • 1% BSA (w/v)
    • Add List Item

    Avidin Mix

    Make fresh.

    • 100 uL avidin buffer
    • 1 uL avidin FITC (Becton Dickinson)
    • 0.5% Triton-X 100 per individual sample
    Note: 5X buffer, 10X CoCl, TdT enzyme and biotin-16-dUTP are from Boehringer Mannheim.

3.5 Detection of apoptotic cells using TUNEL for flow, with surface staining and/or DNA staining optional

  1. Optional, for surface staining: Stain cells using standard surface staining protocol using PE and/or TRI/PERCP color monoclonal antibodies.
  2. Cells are fixed with 2 mL of 1% paraformaldehyde in PBS (pH 7.4) for 15 minutes at 4°C. Wash twice with PBS. Can be stored at 4°C.
  3. Centrifuge sample. Rehydrate pellet in PBS. Centrifuge. Remove supernatant and blot pellet.
  4. Cells are resuspended in 50 µL of cacodylate buffer containing 0.2 M potassium cacodylate, 2.5 mM Tris-HCl (pH 6.6), 2.5 mM CoCl2, 0.25 mg/ml BSA, 7 units of Tdt and 0.5 µM biotin-16-dUTP. Cells are incubated at 37°C for 1 hour. Wash twice with PBS. Remove supernatant and blot pellet. Control tubes are resuspended in buffer without the Tdt enzyme.
  5. Resuspend pellet in 100 µL of buffer made of 4X SSC with 0.1% Triton X-100, 1% BSA and 2.5 µg/mL avidin FITC. Incubate for 30 minutes at room temperature in the dark. Wash cells twice with PBS with 0.1% Triton X-100.
  6. Optional, for DNA: Resuspend cells in 500 µL of PBS. Add 100 µL of Hoechst 33342 stock (10 µg/mL in PBS + 10% Tween 20) to result in a final concentration of 0.5 µg/mL. Incubate at 4°C for 8 hours.

3.6 Labeling apoptosis-induced DNA strand breaks using BrdUTP: An alternative TUNEL method without the use of biotin-dUTP

The TUNEL technique remains the gold standard of detection of apoptosis within cells, although newer methods which detect earlier stages of apoptosis are available. This method is directly equivalent to the TUNEL technique except for the fact that BrdUTP is substituted for biotin-dUTP. BrdUTP is almost three orders of magnitude less expensive than biotin-dUTP and is actually almost a log brighter in intensity than the biotin-dUTP.

  1. After the desired treatment or culture conditions, cells are pelleted and fixed in 1% methanol-free formaldehyde (Polysciences, Inc., Warrington, PA, USA) in Hank's buffered saline solution (HBSS) for 15 minutes on ice. Cells are centrifuged and washed once with HBSS.
  2. The cell pellet is then post-fixed with ice-cold 70% ethanol and stored at -20°C for up to four days.
  3. Cells are rehydrated by washing twice in PBS.
  4. The cell pellet (about 1 x 106) is blotted to remove all extraneous supernatent as possible. The pellet is resuspended in 50 µL of TdT reaction buffer containing: 10 µL of 5X buffer solution (1M potassium cacodylate; 125 mM Tris-HCl, pH 6.6; 1.26 mg/mL bovine serum albumin (BSA); 5 µL of 25 mM cobalt chloride; 0.5 uL (12.5 units) of TdT (all from Boehringer Mannheim, Indianapolis, IN, USA), and 0.25 nmoles of BrdUTP (Sigma) is added.
  5. Cells are incubated at 37°C for 60 minutes. The reaction is stopped by washing the cells twice in 15mM EDTA-NaOH, pH 7.8.
  6. The cells are then incubated in 100 µL of a solution containing 0.7 µg of FITC-conjugated anti-BrdUrd monoclonal antibody (Becton Dickinson, San Jose, CA, USA, clone B44), 0.1% Triton-X-100 (Sigma) and 1% BSA. Cells are washed twice with the above buffer (minus the monoclonal antibody).
  7. Cells are counterstained with 5 µg/mL PI in the presence of RNAase as described (Li et al., 1995, Cytometry 20:172).

Detection of apoptotic cells by flow cytometry using different methods of DNA strand break labeling. Bivariate distributions (isometric contour maps) of DNA content vs. DNA strand break labeling representing exponentially growing HL-60 cells incubated for 3H with 0.15 µM camptothecan which preferentially induces apoptosis (Ap) of cells progressing through S phase (Del Bino et al., 1991). The first three panels to the left represent indirect labeling of DNA strand breaks, utilizing either BrdUTP, digoxygenin conjugated dUTP (d-dUTP) or biotinylated dUTP (b-dUTP). The two right panels show cell distributions following a direct, single-step DNA strand break labeling, either with BDIPY or FITC conjugated dUTP. Note exponential scale of ordinate. As is evident, the greatest separation of apoptotic from non-apoptotic cells is achieved following DNA strand break labeling with BrdUTP. 

References: Labeling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. (1995) X. Li and Z Darzynkiewicz. Cell Proliferation 28:571-579.

3.7 TUNEL on slides (ApopTag® Plus)

TUNEL assay on slides (ApopTag® Plus kit for [1cm x 1.5 cm area])

Fixation

  1. Fix cells using BD lysing solution at 4°C for 10 minutes (1.5 % formaldehyde).
  2. Wash cells 1X in PBS, dry, freeze at -20°C.
  3. Thaw slides at room temperature.
  4. Fix slides in 3:1 mixture of methanol:acetic acid for 5 minutes.
  5. Add about 40 µL Equilibration Buffer (use coverslip). Incubate for at least 30 minutes.
  6. Apply Working Strength TdT (a mixture of 19 µL of Reaction Buffer and 8 µL of TdT Enzyme. Vortex well in a microcentrifuge tube). Incubate slides at 37°C for 1 hour.
  7. Wash slides 3X with PBS for 5 minutes each.

Detection

  1. Add Working Strength Antibody Solution (a mixture of 19 µL Blocking Solution and 16 µL of Anti-Digoxigenen-Fluorescein. Vortex well in a microcentrifuge tube). Incubate slides for one hour in the dark.
  2. Wash 3X with PBS.
  3. Counterstain with DAPI (prepare it by mixing 1 µL/50 mL of stock solution, and adding H2O), coverslip the slides and leave them for 1-2 minutes.
  4. Wash slides with tap water and dry them with air.
  5. Apply one drop of Antifade (Vectashield) on each slide and coverslip them.
  6. Examine slides under microscope using 60X lens.

Contributed by: Dr Katharina Clodi

3.8 Caspase-3 assay by PhiPhi Lux

Description

PhiPhiLux is a heptapeptide substrate for Caspase-3 containing two rhodamines which become fluorescent upon cleavage of the substrate. Emission max=530 nm (Fl1) when excited with a 488-nm laser.

Reagents

  • PhiPhi Lux reagent (OncoImmunin, Inc., College Park, MD) 
  • Phosphate buffered saline (PBS) 
  • Propidium iodide

Procedure

  1. Transfer 3-5 x 105 cells to FACS tube.
  2. Wash cells twice with PBS, and resuspend cell pellet in residual PBS .
  3. Add 5 µl of PhiPhi Lux reagent, and incubate for 1 hr at 37°C.
  4. Wash with cold PBS.
  5. Resuspend cells in 0.5 ml cold PBS, and add 1 µl of 2 mg/ml propidium iodide for live cell gating.
  6. Flow analysis: PhiPhi Lux fluorescence is measured on Fl1. Cells with caspase-3 activity will have a distinctly higher Fl1 fluorescence than those with no activity.

Contributed by: Irina Stiouff and Douglas Weidner

3.9 Mitochondrial membrane potential potential by CMX-Ros (Mito Tracker Red)

Reagents

  • CMX-Ros (Mito Tracker Red, Molecular Probes, Eugene, OR)
  • DMSO
  • Phosphate buffered saline (PBS)

CMX-Ros reagent preparation

The reagent is supplied as a lyophilized powder in 50 µg aliquots. A 1-mM stock is prepared by adding 94 µl DMSO to the lyophilized powder, and stored at -20°C. The stock is diluted to a concentration of 30 nM in RPMI medium warmed to 37°C just before staining.

Procedure

  1. Transfer 3-5 x 105 cells to FACS tube.
  2. Wash cells twice with PBS, and resuspend cell pellet in residual PBS.
  3. Add 300-500 µl of 30 nM CMX-Ros/RPMI at 37°C.
  4. Incubate cells for 1 hr at 37°C.
  5. Pellet cells and wash with cold PBS.
  6. Add 500 µl PBS to final cell pellet, and keep on ice until flow analysis.
  7. Flow analysis: Measure CMX-Ros fluorescence on FL3 (Em max=599 nm). Cells that have disruption of mitochondrial membrane potential appear as a distinct peak with reduced FL3 fluorescence.

Contributed by: Irina Stiouff and Douglas Weidner

Reference: Macho et al., Cytometry 25 (1996), 333

3.10 Mitochondrial membrane potential potential by JC-1

In the presence of high mitochondrial membrane potential, JC-1 forms the so called "J-aggregates" with Fl emission max of 590 nm (Fl 2). When mitochondrial membrane potential is disrupted, as occurs during apoptosis, JC-1 becomes monomeric, and emission max of the monomers is at 525 nm (Fl 1). Thus, a shift of Fl from red to green indicates loss of mitochondrial membrane potential.

Reagents

  • JC-1 (Molecular Probes, Eugene, OR)
  • DMSO
  • Phosphate buffered saline (PBS)

JC-1 reagent preparation

The reagent is supplied in solid form in 5 mg units. A 5 mg/ml stock is prepared by adding 1 ml DMSO to the solid JC-1, and stored at -20°C. The stock is diluted to a working concentration of 5 µg/ml in PBS prewarmed to 37°C just before staining. [5 mg/ml=7.7 mM]

Procedure

  1. Transfer 3-5 x 105 cells to FACS tube.
  2. Wash cells twice with PBS, and resuspend cell pellet in residual PBS.
  3. Add 300-500 µl of 5 µg/ml JC-1.
  4. Incubate cells for 10 min at 37°C.
  5. Pellet cells and wash with cold PBS.
  6. Add 500 µl PBS to final cell pellet, and keep on ice until flow analysis.
  7. Flow analysis: Measure Fl1 and Fl2 fluorescence. Cells that have disruption of mitochondrial membrane potential appear as a distinctly green shifted population when plotting Fl1 vs Fl2.

Contributed by: Douglas Weidner

Reference: Salvioli et al., FEBS Lett 411 (1997) 77-82

4. Proliferation Antigens

4.1 PCNA (Proliferating Cell Nuclear Antigen)

Reagents and Antibodies

  • PBS
  • 100% Methanol
  • Paraformaldehyde
  • Lysolithin (L-alpha lysophosphatidylcholine) (Sigma # L-4129)
  • Nonidet (NP40) (Sigma # N3516)
  • RNAse (Worthington Biochemical Corp # LFO-5679)
  • Bovine Serum Albumin (BSA) Fraction V (Sigma)
  • Propidium iodide (Sigma # P-4170)
  • Anti-PCNA (Boehringer Mannheim Biochemicals # 1170406)
  • Goat-anti-Mouse FITC (GAM-FITC) (Becton Dickinson #9031)
  • MsIgG1 isotype control (100 test) (Coulter #6602872)
  • Paraformaldehyde-Lysolecithin Solution (make fresh):
    • 20-40 µg/mL lysolecithin
    • 1% paraformaldehyde in PBS without Ca and Mg

Procedure

  1. Pellet 1 x 106 cells and wash 2 times in cold PBS. Use 1.5-mL polypropylene Eppendorf tubes.
  2. Incubate pellet in 1 mL paraformaldehyde-lysolecithin solution for 2 minutes at room temperature.
  3. Spin down (4000 rpm for 1.5 minutes), aspirate supernatant and vortex gently to prevent breaking cells.
  4. Incubate pellet with 1 mL of 100% ice-cold methanol for 5 minutes on ice.
  5. Spin down, aspirate supernatant and vortex.
  6. Incubate pellet in 1mL of 0.1% NP40 in PBS without Ca and Mg for 5 minutes on ice.
  7. Spin down, aspirate supernatant and vortex.
  8. Incubate cells with 1 µg (1 µL) of anti-PCNA monoclonal antibody diluted in 100 µL of PBS with 0.1% BSA for 30-60 minutes at room temperature. Use same concentration of IgG1 (5 µL IgG1 in 195 µL PBS) as the isotypic control.
  9. Spin down, aspirate supernatant and vortex gently.
  10. Wash with 0.5 mL PBS with 0.1% BSA.
  11. Add 1000 units (10 µL) RNAse and 0.5 mL propidium iodide (50 µg/mL in PBS).

4.2 Ki-67/DNA

Reagents

  • PBS
  • BSA
  • Nonidet (NP40)
  • 70% methanol
  • Paraformaldehyde
  • RNAse
  • PI

Antibodies

  • MsIgG1 isotypic control, unconjugated (Coulter #6602872)
  • Ki-67 unconjugated (DAKO #M722)
  • Goat-anti-Mouse FITC (GAM-FITC) (Becton Dickinson #9031)

Procedure

  1. Pellet 106 cells, add 1 mL paraformaldehyde (1% in PBS) and incubate 5 minutes at room temperature. Spin down.
  2. Add 1 mL 70% ice-cold methanol, incubate 5 minutes at -20°C. Spin down.
  3. Add 1 mL NP40 (0.1% in PBS), incubate 5 minutes on ice. Spin down, wash twice with PBS.
  4. Incubate cells with 5 µL of KI-67 monoclonal antibody diluted with 50 µL of PBS with 1% BSA for 60 minutes on ice.
  5. Add 10 µL of GAM-FITC diluted with 90 µL PBS/BSA (1%) for 30 minutes on ice. Wash twice with PBS.
  6. Add 500 µL of propidium iodide (50 µg/mL) + RNAse (1000 U/mL), incubate for 1 hour or overnight.
  7. Read flow.

4.3 Surface antigen/KI-67 (Phenotype of Proliferating Cells)

Reagents

  • 3.54 g Na2HPO4 in 100 mL DDW (buffer is 0.2M)
  • 1.37 g NaH2PO4 in 100 mL DDW (buffer is 0.1M)
  • Lysine stock solution:
    • 1.862 g lysine-hydrochloride in 50 mL 0.2M Na2HPO4
    • Adjust pH to 7.4 with 0.1M NaH2PO4
    • Make up to a total volume of 100 mL with DDW
    • Store at 4°C
  • Paraformaldehyde stock solution:
    • 9 mL Na2HPO4, 19 mL NaH2PO4, 22 mL DDW, 4g paraformaldehyde
    • Heat to 60°C, add 2N NaOH until the solution is clear
    • Adjust pH to 7.4
    • Store at 4°C
  • Sodium-meta-periodate (Sigma)

Antibodies

  • IgG1 FITC (Becton Dickinson)
  • KI-67 FITC (DAKO #F788)
  • Any PE-conjugated monoclonal antibody against a surface epitope

Procedure

  1. Prepare PLP-reagent: 7.5 mL of lysine stock solution + 2.5 mL paraformaldehyde stock + 21.4 mg sodium-meta-periodate. This working solution is good for seven days.
  2. Incubate 106 cells with the PE-labeled surface marker (see immunophenotyping protocol). Wash twice with PBS.
  3. To the cell pellet, add 1 mL of PLP-reagent, and incubate for 15 minutes on ice.
  4. Spin cells down and wash twice with PBS.
  5. Add 10 µL KI-67 FITC diluted with 40 µL PBS/BSA (1%). Prepare isotypic control. Incubate for 30 minutes on ice. Wash twice with cold PBS.

4.4 p120 (nuclear antigen)/DNA

Reagents

  • PBS
  • BSA
  • Nonidet (NP40)
  • 70% methanol
  • Paraformaldehyde
  • RNAse
  • Propidium iodide

Antibodies

  • MsIgG1 unconjugated (Coulter #6602872)
  • p120 monoclonal antibody (Coulter)
  • Goat-anti-Mouse FITC (GAM-FITC) (Becton Dickinson)

Procedure

  1. Pellet 106 cells, add 1 mL paraformaldehyde (1% in PBS) and incubate 5 minutes at room temperature. Spin down.
  2. Add 1 mL ice-cold 70% methanol, incubate 5 minutes at -20°C. Spin down.
  3. Add 1 mL NP40 (0.1% in PBS), incubate 5 minutes on ice. Spin down, wash twice with PBS.
  4. Incubate cells with 2 µL of p120 monoclonal antibody diluted with 50 µL of PBS with 1% BSA for 60 minutes on ice. Wash twice with PBS.
  5. Add 10 µL of GAM_FITC diluted with 90 µL PBS/BSA (1%) for 30 minutes on ice. Wash twice with PBS.
  6. Add 500 µL of propidium iodide (50 µg/mL) + RNAse (1000 U/mL), incubate for 1 hour or overnight.

5. Analysis of Oncogene Proteins

5.1 Bcl-2 Oncoprotein

  1. Ficoll bone marrow according to previous protocol.
  2. Adjust the cell concentration to 5 x 104 cells/uL in PBS.
  3. Use 50 µL of this cell suspension for the following steps, or multiply all mentioned amounts with an appropriate number.
  4. Incubate 30 minutes on ice with antibodies to surface antigens: Isotype controls are prepared the same way as for normal immunophenotyping.
  5. Wash twice with 2 mL of PBS.
  6. Resuspend the pellet in 1 mL PBS, vortex gently.
  7. Add 1 mL of 2% ice-cold paraformaldehyde in PBS while vortexing the sample (final concentration 1%). Incubate on ice for 1 hour. Wash twice with PBS.
  8. Resuspend the pellet in 1.9 mL of PBS. Add 0.1 mL of 10 % Tween-20 in normal saline, vortex gently. Incubate on ice for 15 minutes.
  9. Wash twice in PBS.
  10. Add 10 µL of bcl-2 FITC (DAKO) antibody in 50-90 µL of cell suspension, and incubate on ice for 15 minutes. Also add 10 µL of isotype control to the appropriate tube.
  11. Wash twice in normal saline and resuspend in 1.9 mL of normal saline.
  12. Incubate for 30 minutes on ice.
  13. Add 0.1 mL Tween-20 10% in normal saline, vortex gently. Add 60 µL of Hoechst 33343 stock solution (100 µg/mL in normal saline). Incubate overnight in the refrigerator, and run the sample in the morning.

5.2 p21 ras (+DNA)

  1. Start with 1.5 x 106 leukocytes in a 3.7-mL conical bottomed plastic test tube without supernatant.
  2. Fix the cells in 1 mL of 100% ice-cold methanol, adding to the cells while vortexing gently. Continue to vortex until the pellet disintegrates.
  3. Freeze the cells at -20°C for exactly 10 minutes.
  4. Add 50 µL of PBS without Ca, Mg, with 1% BSA and 1% whole goat serum. Let sit at room temperature for 30 minutes. Vortex occasionally.
  5. Add 50 µL of anti-Ras antibody diluted 1:98 in PBS with 1% BSA and 1% whole goat serum. Incubate on ice for 30 minutes.
  6. Wash twice with PBS, leaving approximately 50 µL of supernatant remaining.
  7. Add 50 µL of rabbit-anti-rat antibody diluted 1:10 with PBS without Ca, Mg, +1% BSA, +1% whole goat serum. Vortex and incubate on ice for 30 minutes.
  8. Wash twice with 1 mL PBS, leaving approximately 50 µL of supernatant.
  9. Add 50 µL of Cappel GAM-FITC diluted 1:40 with PBS without Ca, Mg, +1% BSA, +1% whole goat serum. Incubate on ice in the dark for 30 minutes.
  10. Wash twice with 1 mL PBS.
  11. Add 250 µL of 500 U/mL highly purified RNAse. Incubate in the dark at 37°C for 30 minutes.
  12. Add 250 µL propidium iodide (Sigma) directly to tube.
  13. Cells can be stored at 4°C until measuring.

6. Multidrug Resistance Assays

6.1 Detection of P-glycoprotein using the antibody MRK16

MDR1 Expression

Single color

  1. 1 x 106 cells are resuspended in 100 µL of PBS with 2 µL of MRK16 (Kamiya, Thousand Oaks, CA) (mouse IgG2a; 1.0 µg total MoAb). Pure mouse IgG2a (Caltag) used at same concentration for control (10 µL antibody in 100 µL cell suspension). Incubate cells 20 minutes on ice. Wash once with cold PBS, centrifuging at 330 x g for 8 minutes.
  2. The cell pellet is resuspended in 100 µL of PBS with 2 µL Biotin-Goat anti Mouse IgG2a antibody (Southern Biotech, Birmingham, AL) (1 µg total MoAb). Incubate cells 20 minutes on ice. Wash once with cold PBS.
  3. Resuspend cell pellet in 100 µL PBS with 4 µL streptavidin conjugated to RED613 (Becton Dickinson, San Jose, CA) 20 minutes on ice. Wash once with cold PBS.
  4. Read protein expression by flow on FL3 on a Becton Dickinson FACScan (excitation at 488, emission at 613).

Multicolor

  1. 1 x 106 cells are resuspended in 100 µL of PBS with 2 µL of MRK16 (Kamiya, Thousand Oaks, CA) (mouse IgG2a; 1.0 µg total MoAb). Pure mouse IgG2a (Caltag) used at same concentration for control (10 µL antibody in 100 µL cell suspension). Incubate cells 20 minutes on ice. Wash once with cold PBS, centrifuging at 330 x g for 8 minutes.
  2. The cell pellet is resuspended in 100 µL of PBS with 2 µL Biotin-Goat anti Mouse IgG2a antibody (Southern Biotech, Birmingham, AL) (1 µg total MoAb). Incubate cells 20 minutes on ice. Wash once with cold PBS.
  3. Resuspend cells in either pure goat serum for 10 minutes for nonspecific binding, or 100 µL PBS with 5 µL IgG2a (0.5 µg MoAb). Wash 1X cold PBS.
  4. Resuspend cell pellet in 100 µL PBS with streptavidin conjugated to RED613 (Becton Dickinson, San Jose, CA) and desired PE and FITC conjugated antibodies. Incubate for 20 minutes on ice. Wash once with cold PBS.
  5. Read samples directly by flow.

Notes: To analyze samples, the histogram of MRK16 expression is overlaid on that of the control. Differences in fluorescence are assessed using the Kolmogorov-Smirnov (KS) statistic, denoted as D, which measures the difference between two distribution functions and generates a value ranging from -1.0 to 1.0. This method accurately identifies small differences in fluorescence and is useful in detection of low level MDR1 expression. MRK16 staining intensity is categorized as follows: negative (d < 0.10), dim (0.10 < D < 0.15), moderate (0.15 < D > 0.25) and bright (D > 0.25).

References:

  • C.P. Leith et al. (1995) Correlation of Multidrug Resistance (MDR1) Protein Expression with Functional Dye/Drug Efflux in Acute Myeloid Leukemia by Multiparameter Flow Cytometry: Identification of Discordant MDR1-/Efflux+ and MDR1+/Efflux- Cases. Blood 86(6) 2329-2342.
  • C.P. Leith et al. (1997) Acute Myeloid Leukemia in the Elderly: Assessment of Multidrug Resistance (MDR1) and Cytogenetics Distinguishes Biologic Subgroups with Remarkably Distinct Responses to Standard Chemotherapy. Blood 89(9) 3323-3329.

6.2 Detection of p-glycoprotein using 4E3-FITC (Signet)

  1. Resuspend about 1 x 106 cells in 100 µL of PBS. Add 5 µL of IgG2a-FITC to isotypic control tube and 5 µL of 4E3-FITC to sample tube.
  2. Incubate for 15-30 minutes on ice.
  3. Wash with 2 mL cold PBS on time.
  4. Resuspend pellet in 200-300 µL cold PBS for FACS analysis.

Notes: An isotypic control must always be run with the 4E3 so that Kolmogorov-Smrinov statistics can be done as with the MRK16 (see above). A D-value of greater than 0.15 is considered positive. Multicolor flow may easily be done with the 4E3-FITC.

6.3 Anthracycline uptake (Daunorubicin): Functional assay

  1. Adjust cells concentration to 1 x 106 cells/mL in RPMI + 10% FCS at 37°C.
  2. Take 500 µL for the start point and add 500 µL fresh warm medium to the original tube.
  3. Incubate 2 mL of the above cell solution under the following conditions:

    5 µg/mL DNR
    5 µg/mL DNR + 10 µg/mL verapamil
    10 µg/mL verapamil

  4. Take 500 µL at different timepoints. Centrifuge, resuspend pellet in cold PBS. Wash 1X, resuspend pellet in 250 µL cold PBS.
  5. Read flow immediately.

6.4 Rhodamine-123 efflux: Functional assay

  1. 1 x 106 cells are preincubated with verapamil (10 µg/mL) and rhodamine-123 (0.5 mg/mL) in RPMI + 10% FCS for 37°C.
  2. Wash cells twice with medium.
  3. Resuspend cells in medium without rhodamine-123 in the presence or absence of mdr inhibitors such as verapamil (10 µg/mL).
  4. An aliquot is measured immediately, following measurements are performed every 15 minutes.

Double staining with surface markers (PE- and PERCP-conjugated) is possible and allows the correlation with certain cell populations. If this is desired, staining for surface markers should be done before dye efflux.

6.5 DiO(C2)3 MDR functional efflux

  1. Suspend cells in 10 mL of 37°C medium containing DiO(C2)3 at a final concentration of 60 ng/mL. Incubate sample in a 37°C waterbath for 30 minutes. Spin down at 4°C. Resuspend pellet in 3 mL of fresh, warm, dye-free medium. Divide sample into three aliquots. Place one sample on ice to be read immediately by flow for baseline uptake.
  2. Add warm, dye-free medium up to 3 mL in remaining two aliquots. Add cyclosporin A (CsA, Sandoz Pharmaceuticals, Basel, Switzerland) at a final conc of 2.5 µg/mL, or PSC833 (Sandoz Pharmaceuticals) at a final concentration of 1 µg/mL to one tube to inhibit the p-glycoprotein pump. Incubate at 37°C in a water bath for 90 minutes. Wash cells with medium at 4°C and resuspend pellet in 4°C medium. Store on ice for flow.
  3. For multicolor analysis, cells may be stained with desired PE- and PERCP/TRI-conjugated antibodies and washed in medium at 4°C.

Notes: DiOC2 is excited at a wavelength of 488 nm with emission at 500 nm, therefore flow is read on FL1 on a Becton Dickinson FACScan. DiOC2 is superior to Rhodamine-123 (RH123) for functional analysis of p-glycoprotein (p-gp) due to a tighter peak and due to the fact that it is only pumped by p-gp, whereas RH123 is pumped by other multidrug resistance proteins. When using single color analysis of protein function, propidium iodide may be added to the sample at a final concentration of 5 µg/mL 10 minutes prior to flow to distinguish dead from live cells. Efflux is assessed by analyzing differences in fluorescence of the overlaid histograms of cells in the presence/absence of p-gp inhibitor using the Kolmogorov-Smirnov (KS) statistic. A D value of > 0.25 is used to define a case as efflux positive.

References:

  • C.P. Leith et al. (1995) Correlation of Multidrug Resistance (MDR1) Protein Expression with Functional Dye/Drug Efflux in Acute Myeloid Leukemia by Multiparameter Flow Cytometry: Identification of Discordant MDR1-/Efflux+ and MDR1+/Efflux- Cases. Blood 86(6) 2329-2342.
  • C.P. Leith et al. (1997) Acute Myeloid Leukemia in the Elderly: Assessment of Multidrug Resistance (MDR1) and Cytogenetics Distinguishes Biologic Subgroups with Remarkably Distinct Responses to Standard Chemotherapy. Blood 89(9) 3323-3329.

7. Cytokine Receptors

7.1 Fluoresceinated/biotinylated cytokines

This assay is based on the principle that cytokines act as specific ligands for their receptor on the surface of target cells.

Reagents

  • Fluorokines (directly conjugated fluorochromes or biotin-conjugated) from R&D Systems, includes Fluorokine washing solution.
  • Unlabeled cytokines for blocking experiment

Procedure

  1. 1e5 cells in a volume 25 µL are incubated with 10 µL of conjugated cytokine for 60 minutes on ice. Shake well after adding the Fluorokine.
  2. Wash twice with Fluorokine washing solution. (made up with stock solution diluted 1:10 with PBS).
  3. When using biotinylated Fluorokine, add 10 µL of streptavidin-PE, incubate for 60 minutes on ice.
  4. Wash twice as in step 2.

As a control, unlabeled cytokine is used in 10 M excess of the fluoresceinated cytokine: Start with the same amount of cells, add the unlabeled cytokine, shake for a week, incubate for 15 minutes on ice, add the Fluorokine and proceed as above.

Double labeling

The availability of fluoresceinated/biotinylated cytokine offers the possibility to double stain with other surface markers. Double staining follows the protocol as under Bcl-2 Oncoprotein. In addition, counterstaining with propidium iodide can be performed.

7.2 Receptor Antibodies

This method follows the principles of surface marker staining. As an example, labeling with an antibody against the human IL-3 receptor is described.

Reagents

  • PBS
  • BSA
  • IL-3 receptor antibody (provided by DNAX)
  • IgG1 isotypic control antibody (Coulter)
  • Goat-anti-Mouse FITC (GAM-FITC) (Becton Dickinson)

Procedure

  1. 2e5 cells + IL-3 receptor antibody, added to a final concentration of 5 µg/mL. Incubate for 30 minutes on ice.
  2. Wash twice with PBS.
  3. Add the secondary antibody: 5 µL of GAM-FITC diluted with 45 µL of PBS-BSA (1%), incubate for 30 minutes on ice.
  4. Wash two times with PBS.

8. Other Flow Cytometric Assays

8.1 Reporter gene assay: beta-galactosidase measurement

FDG= fluorescein-di-beta-galactopyranoside

Dissolve 5 mg in 3.8 mL DDW= 2 mM FDG.

  1. Prepare cells in medium (alpha-MEM + 10% FCS + antibiotics), adjust to a concentration of 107 cells/mL.
  2. Place 100 µL of the cell suspension in a FACS-tube, incubate in 37°C water bath for 5 minutes.
  3. Add 100 µL of 2 mM FDG, prewarmed to 37°C, mix and incubate again for 1 minute at 37°C.
  4. Place on ice, add 1800 µL of ice-cold isotonic medium (alpha-MEM + FCS+ antibiotics); incubate for 60 minutes on ice.

9. Magnetic Cell Sorting

9.1 Separation of CD34+ cells from peripheral blood, bone marrow or pheresis

9.1a Separation of normal CD34+ cells from frozen bone marrow

  1. For normal bone marrow frozen in bags:

    Place bag into a plastic zip-lock bag. Place into a 37°C water bath and rapidly thaw cells until only just slushy.

    Wipe the port area of the bag well with 70% ethanol and insert a sterile port adapter.

    As gently and as quickly as possible, withdraw the cell suspension from the bag using a 30- or 60-cc syringe with at least a 18-gauge needle, preferably 16-gauge.

    Remove needle from syringe and transfer 20 mL cell suspension to 50 mL conical tubes containing 30 mL warm (37°C) buffer while gently swirling. Centrifuge immediately.

  2. For normal bone marrow frozen in tubes:

    Work in batches of 4-5 (5-mL) vials. Place vials into plastic zip-lock bag. Place bag into 37°C water bath and rapidly thaw cells until just slushy.

    Wipe the tubes well with 70% ethanol.

    Transfer the contents of 4-5 vials to a 50-mL conical tube containing 30 mL warm buffer while gently swirling. Centrifuge immediately.

  3. Remove supernatant from tubes carefully as pellet may not be tightly packed. Gently scrape the tube to loosen pellet. Fill each tube to 50 mL warm buffer. Vortex gently to mix cells well with buffer. Centrifuge.
  4. Repeat step 3, but combine cells from two tubes into one.
  5. Resuspend pellet in 40 mL warm buffer. Vortex gently to mix cells. If there is a nice single cell suspension, the cells may be Ficolled to remove dead cells and granulocytes. If there are visible "strings" of cells, the suspension needs to be incubated at 37°C while gently shaking and then centrifuged. This needs to be repeated until a single cell suspension is achieved.
  6. To Ficoll, layer 20 mL of the cell suspension over 15 mL of room temperature Ficoll. Centrifuge at 1800 rpm for 15-20 minutes.
  7. Remove mononuclear layer of cells from Ficoll. Wash cells with cold buffer in 50-mL conical tubes. Centrifuge.
  8. Combine all tubes to one 50-mL conical tube. Fill to 50 mL with cold buffer. Remove a small sample (less than 100 µL) to count cells. Centrifuge.
  9. Remove supernatant. Scrape pellet gently. Based on total cell count, add ? the amount of cold buffer recommended by Miltenyi. Then add ? the recommended amount of antibody (solution A1 and solution A2 for the indirect MACS kit). Vortex gently to mix cells and antibody well.
  10. Incubate cells in the refrigerator for 15 minutes. Fill tube to 50 mL cold buffer and centrifuge. Wash a second time with 50 mL cold buffer.
  11. Remove supernatant. Scrape pellet gently. Based on total cell count, add the amount of cold recommended by Miltenyi. Then add ? the recommended amount of antibody (solution B for the indirect MACS kit). Vortex gently to mix cells and antibody well.
  12. Incubate cells in the refrigerator for 15 minutes. Fill tube to 50 mL cold buffer and centrifuge.
  13. Remove supernatant. Scrape pellet gently. Add enough cold buffer to the cells to give a final cell concentration of about 1x108 cells/mL for VS column and 1x107 for RS column.
  14. Load column with cold degassed buffer.
  15. Filter the cell suspension over a 30-um nylon mesh filter. Retain a small amount of cell suspension and record exact volume for quality assurance and quality control (QA/QC).
  16. Add cell suspension. Proceed with separation according to Miltenyi's instructions. Place positive fraction over a second column if a higher purity is desired.
  17. Retain a small amount of the final fraction and record the exact volume for QA/QC.
  • MACS buffer

    • Hank's Balanced Saline Solution (HBSS) -Ca+2, -Mg+2
    • 0.5% BSA
    • 0.6% Anticoagulant Citrate Dextrose - Formula A (ACD-A) (Baxter)
    • 100 U/mL Heparin
    • 100-200 U/mL DNAse ( must be added directly before use)
    • Filter sterilize and store at 4°C

Notes: The most important task in separating frozen marrow is achieving a single cell suspension. This is accomplished mainly with the DNase in the buffer. To that end, you may want to have 200 U/mL DNase in the buffer up to the Ficoll step. You may reduce the concentration to 100 U/mL after removing the mononuclear layer. I usually use 100 U/mL throughout the entire procedure. As well, if you find that you have a particularly hard sample, you may increase the DNase to 200 U/mL, but not higher. It is very important that the cells remain cold after the Ficoll. Ideally the cells would be cold throughout the entire procedure to preserve cell viability, but the necessity of the DNase preempts this.

See Miltenyi insert for information from the company.

9.1b Separation of normal CD34+ cells from fresh pheresis of mobilized stem cells

  1. Resuspend sample up to 100 mL of MACS buffer (see recipe below). Aliquot to two 50-mL conical tubes. Centrifuge for 10 minutes at 1000 rpm to "soft spin" the pellet. A soft spin keeps the platelets, which are concentrated in pheresis samples, in the supernatant. Platelets cause major problems with the staining of the sample as well as the running of the sample through the magnetic column. The supernantant must be aspirated, not poured off, since the pellet is loose.
  2. If the supernatant from the soft spin is still relatively cloudy, the soft spin may be repeated.
  3. ACD-A changes the density of the cells in the pheresis sample so that granulocytes will stay at the interphase of the Ficoll. Although pheresis samples have a high concentration of granulocytes, it is more important to remove the platelets before they activate. An alternative protocol may be that the sample is initially suspended in buffer without ACD-A and then Ficolled. The mononuclear layer may then be resuspended in buffer with ACD-A, and the soft spin performed. We have found it best to just leave the granulocytes and adjust concentration of the antibody (follows).
  4. Count the total number of cells. Combine sample into one 50-mL conical tube. Miltenyi lists antibody amount according to the total number of cells; however, this may be adjusted according to the estimate of number of cells positive for the sorting parameter. Since pheresis has between 1-10% CD34+ cells, we usually use 50% of the recommended amount of antibody. If a Ficoll is not performed, this may be reduced to ? the amount of antibody, but the buffer should not go below ?, and the incubation time should be extended to 30 minutes. Add ? the amount of buffer recommended by Miltenyi. Add ? the amount of reagent A1, shake gently. Add ? the recommended amount of reagent A2, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.
  5. Wash the sample two times with 50 mL of MACS buffer.
  6. Resuspend the sample in _ the recommended amount of buffer, and _ the amount of reagent B, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.
  7. Wash the sample one time with 50 mL of MACS buffer.
  8. Resuspend the sample in at least 10 mL degassed (see Note) MACS buffer for 1 x 109 total cells, or up to 20 mL for 2 x 109 total cells. Run sample over a VS positive selection column.
  9. Wash 2X with 3 mL of degassed buffer.
  10. Attach stop cock and syringe to bottom of VS column. Remove column from magnet. Backflush column with 6 mL of degassed buffer. Replace column in magnet.
  11. Remove stop cock. Allow buffer to flow through column. Wash 2X with 3 mL of buffer.
  12. Remove column from magnet. Add 6 mL buffer to column and allow to run through. Add 6 mL buffer to column and plunge the column.
  13. Count the total number of cells collected from each fraction to calculate the recovery of the separation.
  14. Perform flow cytometry on the collected fractions to assess sample purity with CD45-FITC and CD34-PE (Becton Dickinson).

    MACS buffer

    • Hank's Balanced Saline Solution (HBSS) -Ca+2, -Mg+2
    • 0.5% BSA
    • 0.6% Anticoagulant Citrate Dextrose- Formula A (ACDA) (Baxter)
    • Filter sterilize and store at 4°C

Notes: It is very important when running the magnetic column that only degassed MACS buffer be used. To degas the buffer, place 100 mL of buffer in a 150-mL bottle. Place a rubber stopper attached to a vacuum line over the mouth of the bottle. Turn vacuum on. Allow the buffer to degas at room temperature for at least 30 minutes. Replace cap on bottle and refrigerate buffer until cold. Use buffer as directed.

10. Fluorescence in Situ Hybridization Techniques

10.1 In situ hybridization with centromeric chromosome 7 and chromosome 3 on suspension from tissue preparations

  1. Cells are fixed 2X in methanol: acetic acid, 3:1 and dropped on a slide. Pretreatment of slides is very important for cells from lymph nodes or other tissue preparations.

    2 minutes 2X SSC
    5 minutes Triton-X/ HCl
    5 minutes PBS
    5 minutes PBS/MgCl2 (% mL 1M MgCl2 + 95 mL PBS)
    5 minutes Formaldehyde 1% (1.35 mL 37% Formaldehyde + 48.7 mL MgCl2/PBS)
    5 minutes PBS
    Ethanol series (70%, 85%, 100%)
    Air dry slides

  2. Probe cocktail: 7 µL CEP Buffer (Imagenetics comes with centromeric chromosomes)

    1 µL centromeric chromosome 7 probe
    2 µL centromeric chromosome 3 (Cy3) (Oncor)

    Vortex well. Denature at 73oC for 5 minutes.

  3. Heat Coplin Jar with 70% formamide/2X SSC to 37°C. Denature slide for 3 minutes. Put slides immediately in ice cold alcohol series 3 minutes each (70%, 85%, 100%).
  4. Hybridize overnight.
  5. Wash 3X 5 minutes in 65% formamide/2X SSC at 37°C inside Coplin Jar.

    1X 8 minutes 2X SSC at 37°C
    1X 5 minutes 4X SSC/Tween 20

  6. Counterstain with DAPI 1 mL on each slide for 1 to 2 minutes. Wash gently with normal H2O and dry with air.
  7. Add 1 drop of Antifade (Vectashield) and add coverslip.

    Hybridize two nights.

  8. Wash 3X 5 minutes 50% formamide/2X SSC at 42°C inside Coplin Jar.

    3X 5 minutes 0.1X SSC at 60oC
    Blocking of unspecific binding in 4X SSC/0.2% Tween 20 + 5% BSA
    1:200 avidin-FITC (Vector Antibodies)
    1:200 anti-avidin biotinylated

  9. All antibodies are diluted in 4X SSC/Tween 20/BSA.

    Avidin FITC for 30 minutes

  10. Wash 3X 5 minutes 4X SSC/Tween 20

    Anti-Avidin FITC for 45 minutes

  11. Wash 3X 5 minutes 4X SSC/Tween 20

    Avidin-FITC for 30 min

  12. Wash 3X 5 minutes 4X SSC/Tween 20
  13. Counterstain with DAPI 1 mL on each slide for 1 to 2 minutes, wash gently with normal H2O and dry with air.
  14. Add 1 drop of Antifade (Vectashield) and add coverslip.

11. PCR Methods

11.1 PCR methods for MDR1

PCR conditions

1.0 µg of total RNA in 10 µL reaction is used for reverse transcription. One twentieth of the reaction is used for PCR reaction. The total volume of the PCR reaction is 25 µL. The final concentration of the biotin-labeled MDR1 A, B2-M A primer and TBR-labeled MDR1 B, B2-M B primer is 0.2 µM, 0.2 M of dNTP, 2.5 µL 10X reaction buffer and 1.5 U.

  1. Spin centrifuge cells at 200 x g. Wash two times with PBS.
  2. Resuspend pellet in 1.5 mL PBS. Add 0.5 mL of 4% paraformaldehyde in PBS while vortexing gently. Incubate at 4°C for 15 minutes.
  3. Wash cells twice with PBS.
  4. Store cells in PBS at 4°C for up to one month.

Phosphate Buffered Saline (PBS) (1X)

  • 0.23 g NaH2PO4 (anhydrous, 1.9 mM)
  • 1.24 g Na2HPO4 (anhydrous, 8.1 mM)
  • 9.25 g NaCl (154 mM)

    Add H2O to 900 mL

    If needed, adjust to desired pH (usually 7.2 to 7.4) with 1M NaOH or 1 M HCl. Add H2O to 1 liter. Filter sterilize if desired. Store indefinitely at 4°C
  • 175.3 g NaCl
  • 88.24 g sodium citrate

    Add 950 mL H2O

    Adjust pH to 7 with 5 M HCl

    Dilute to 1 liter with H2O

    Store <1 month at room temperature

EDTA (ethylenediamine tetraacetic acid) 0.5M, pH 8.0

Dissolve 186.1 g Na2-EDTA.2H2O in 700 mL water with stirring. Adjust the pH to 8.0 with 10 M NaOH, then adjust volume to 1 liter with H2O, and autoclave. Store <1 year at room temperature.

The EDTA will not dissolve fully until the pH is adjusted to 8.


© 2014 The University of Texas MD Anderson Cancer Center