Use of functional dyes as well as antibody-coupled fluorophores provides indispensable tools for the study of all aspects of cell biology including protein expression, cell proliferation and differentiation, cell signaling pathways, enzyme activity, gene regulation, cell lineage, apoptosis, autophagy, and chemotherapeutic resistance. Current instrumentation allows simultaneous quantitative analysis of up to 12 parameters, each of which is assayed at the individual cell level, facilitating high content analysis of mixed cell populations and rare cell types. The Core facility provides researchers with technical expertise in assay development, data acquisition and various data analysis techniques. Core personnel are continuously working with clients to develop new assays or multiplex existing assays to satisfy user research goals. Examples of routine and recently developed assays include:
- Up to 6-color immunophenotype determination of Hoechst 33342-extruding side population (SP) stem cells in leukemias and solid tumors
- Characterization of immune responses by multiplexed intracellular cytokine staining
- MHC/peptide tetramer staining for immune reactivity of lymphoid cell populations
- Live/dead discrimination by amine reactive dyes allowing fixation of biohazardous samples
- Cell cycle and DNA ploidy analysis: EB, DAPI, BrdU, Hoechst, DyeCycle, Ki67, PCNA, AO, nucleolar antigens, cyclins and p-histone H3
- Ca++ mobilization
- Apoptosis: TUNEL, subG1, Annexin V, caspase activation, mitochondrial membrane potential (CMXRos, TMRM, JC-1), Bcl-2, Bax, Bcl-X, BAG-1, Fas, etc.
- Cell division history: CFSE and PKH26 allow determination of the number of actual cell divisions
- Transgene detection: CFP, GFP, YFP, RFP and beta-gal
- Measurement of histone acetylation and protein phosphorylation: p-Erk1/2, p-Stat 1, p-Stat 3, p-Stat 5, p-Stat 6 and p-Akt
- Intracellular cytokine staining
- MHC/peptide tetramer staining
- Oncogenes: p53, c-myc, p21 and ras
- 9-color immunophenotyping (in combination with many of the above)
Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on samples at single-cell resolution utilizing metal labeled antisera. While similar to traditional fluorescence based flow cytometry the use of metal labels removes the need for compensation and alleviates the limitations on the detectible number of parameters. Mass cytometry employs antibodies covalently labeled with non-radioactive isotopes of the lanthanide and noble metals as tags to perform massively multiparametric flow cytometry in a quantitative manner. Currently the system can employ approximately 40 parameters per cell with a theoretical limit of 100. A sample is injected into high-temperature plasma obtained by heating a flowing argon gas stream with radio frequency (RF) energy. Under conditions approximating the surface of the sun, the sample is vaporized, atomized, and ionized as it flows through the plasma. High speed mass analysis provides a "mass fingerprint" that identifies the elements contained in the sample. The attributes of note include: wide linear dynamic range (up to 9 orders of magnitude), exceptional sensitivity (sub-part per trillion, or attomole/microlitre, detection), enormous abundance sensitivity (<10-4 overlap between adjacent isotopes), counting-statistics-limited precision, absolute quantification, and tolerance of concomitant matrix.
The information derived from multiparameter analysis is complemented by the ability to isolate any desired cell population with high-speed cell sorting for culture and further characterization by molecular biology and biochemical techniques. Examples of sorting applications include:
- Hematopoietic progenitor and stem cell sorting (e.g. CD34+/38-, CD34+/38+, CD34+38-123+, Lin-/Sca1+/ c-kit+, Hoechst 33342 SP cells, Aldefluor)
- Sorting Memory/Naïve lymphocyte subsets based on CD45RA/CCR7 expression
- Sorting of regulatory T-cells based on CD127/CD25 expression
- All assays listed above can be adapted and used in combination for cell sorting
Laser Scanning Cytometry
A slide-based system with fluorescence detection and quantitation that can analyze up to three fluorochromes from cells deposited on slides. FACS sorted, adherent or rare tumor or stem cells can be analyzed using this technology. Protein expression data can be obtained from as few as several hundred cells.
Laser Scanning and Spinning Disk systems allow the localization and co-localization of cellular and sub-cellular components and cellular interactions in both fixed and live specimens. The systems are particularly useful in observing multicellular structures from tissues or cultured specimens in their native 3D forms. The Core facility offers researchers tools and techniques for image acquisition, 3D-reconstruction and time-series observation as well as a variety of image processing and analysis functions. Both systems are configured to allow longitudinal analysis of live cells.
The NC facility has an epi-fluorescence microscope outfitted with a CRi Nuance multispectral imaging camera for fluorescence or brightfield imaging. This camera allows for the capture of lambda stacks, series of images across emission wavelengths. By capturing the complete emission spectra a more complete and accurate analysis can be performed. This system is capable of performing these tasks through the use of a LCD tunable filter. The system is ideal for eliminating autofluorescence and quantifying various signals.
|MD Anderson Users||Flow Cytometry Analysis||$50||$70|
|Live Cell Imaging||$300|
|Imaging Mass Cytometry||$50|
|Metal Antibody Labeling||$85||$160|
|Non-MD Anderson Users||Flow Cytometry Analysis||$71.43||$100.00|
|Live Cell Imagine||$428.57|
|Imaging Mass Cytometry||$71.43|
|Metal Antibody Labeling||$121.43||$228.57|