Protocol for Flow Cytometry Cell Surface Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 15 mL conical tube and centrifuge for 4 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.
Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.
Cell surface staining
1. Recommended: Block non-specific interactions using 0.5-1 ug of a species specific Fc-blocking reagent such as an anti-mouse CD16/CD32 antibody (NBP1-27946).
2. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined) to 100 uL of staining buffer (NBP2-26247) per sample (eg. use 1 mL of staining buffer for 10 samples).
3. Mix well and incubate at room temperature in dark for 20 minutes.
4. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
5. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
6. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
7. Incubate at room temperature in dark for 20 minutes.
8. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.
