Flow Cytometry Protocol

NOTE: To simultaneously assess intracellular signaling molecules together with cell surface markers, please use our alternate flow cytometry protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in 800 mL dH₂O. Adjust the pH to 7.4 with HCl and the volume to 1L. Store at room temperature.
2. Formaldehyde (methanol free).
3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.

B. Fixation

1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
3. Fix for 10 minutes at 37°C.
4. Chill tubes on ice for 1 minute.
5. For extracellular staining with antibodies that do not require permeabilization, proceed to step D1 or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization step C1.

C. Permeabilization

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, to remove fix prior to permeabilization, pellet cells by centrifugation and resuspend in 90% methanol.
2. Incubate 30 minutes on ice.
3. Proceed with staining or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

1. Aliquot 0.5-1x10⁶ cells into each assay tube (by volume).
2. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
4. Block in Incubation Buffer for 10 minutes at room temperature.
5. Add the unconjugated, biotinylated, or fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet for the appropriate dilution).
6. Incubate for 1 hour at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml PBS and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to step D9.
9. Resuspend cells in fluorochrome-conjugated secondary antibody* or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.
10. Incubate for 30 minutes at room temperature.
11. Rinse as before in Incubation Buffer by centrifugation.
12. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step E1.

E. Optional DNA Stain

1. Resuspend cells in 0.5 ml of DNA dye (e.g. DRAQ5^® #4084).
2. Incubate for at least 5 minutes at room temperature.
3. Analyze cells in DNA stain on flow cytometer.

*Recommended Secondary Antibodies:

Anti-Rabbit

• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414

Anti-Mouse

• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4409
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410

Anti-Rat

• Anti-Rat IgG (H+L), (Alexa Fluor^® 488 Conjugate) #4416
• Anti-Rat IgG (H+L), (Alexa Fluor^® 555 Conjugate) #4417
• Anti-Rat IgG (H+L), (Alexa Fluor^® 647 Conjugate) #4418