South Campus Flow Core ImageStreamX Mark II Specifications
4 Lasers: 405nm, 488nm, 561nm, 642nm
20x Field of View: 120x256 μm; Imaging Rate: 4,000 cells/sec
40x Field of View: 60x128 μm; Imaging Rate: 2,000 cells/sec
60x Field of View: 40x170 μm; Imaging Rate: 1,200 cells/sec
96 Well Plates
|2||488||505-560 (527/65)||FITC, AF488, GFP, YFP|
|3||561||560-595 (577/35)||PE, AF546, Cy3, PKH26, DSRed|
|4||595-640 (610/30)||PE-TxRed, ECD, AS568, AF610, 7AAD, PI, mCherry, mKate, mTomato|
|5||640-745 (702/86)||PE-Cy5, PerCP, PerCP-Cy5.5|
|6||745-780 (762/35)||SSC, PE-Cy7, Draq5|
|7||405||435-505 (470/70)||DAPI, Hoechst, PacBlue, AF405, eFluor405, CFP, Aqua|
|8||505-570 (537/65)||PacOrange, Cascade Yellow, AF430|
|10||640||595-640 (610/30)||Qdot625, eFluor625|
|11||642-745 (702/86)||APC, AF647, AF660, Cy5, Draq5|
|12||745-780 (762/35)||SSC, APC-Cy7, APC-H7, Cy7, AF750|
Final Sample Concentration and Volume:
At least 1 million cells in 50 µL (2x107 cells/ml) in PBS/2%FBS in a 1.5mL siliconized microcentrifuge tube.
FAQ: What is the minimum number of cells per sample?
The ISX analyzes more than half the 50 µL sample volume, so samples with as few as 10,000 cells will still yield images of over 5,000 cells. The imaging rate is proportional to the sample concentration and assumes a cell concentration of ~2x10^7 cells/ml so dilute samples will have a MUCH LOWER FLOW RATE. Keep this mind when reserving time for your experiment!
Choice of Fluorochrome and Brightness of Stain and Stain Balancing:
Choose traditional cytometry/imaging fluorochromes excited by the available lasers. Quantifying the location and distribution of signals in an image is a demanding task that requires optimized labeling. Below are a few suggestions to help design the experiment:
• Try to achieve at least a full log shift in fluorescence, as measured by FACS
• Use the brightest dye for the antigen with the smallest copy number
• The brightness of probes can be independently controlled by changing the laser power
However, data quality is enhanced when the brightness levels of all probes excited off a single laser are balanced to within a log of each other. Probe balancing avoids the saturation of bright stains when they are combined with dim stains in the same sample.
Samples labeled with a single-color positive control for each fluorochrome used (i.e. FITC only cells, PE only cells) are needed. DO NOT add a nuclear dye (i.e. Hoechst or DAPI) to single color controls.
Minimize aggregation problems by straining the sample through a 70µm nylon mesh strainer, or by using an anti-clumping buffer. Side note: the narrowest pathway in the system is 250µm and all fluids are pumped via motor driven syringe pumps so clogging problems are rare.