Flow Cytometry Live Cell Protocol

A. Solutions and Reagents

• 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 distilled water (dH₂O). Adjust the pH to 7.4 with HCl and the volume to 1 liter with dH₂O. Store at room temperature.
• Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.

B. Immunostaining

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

1. Collect cells by centrifugation and aspirate supernatant.
2. Aliquot 0.5–1x10⁶ cells into each assay tube (by volume).
3. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
4. Resuspend cells in 100 µl Incubation Buffer per assay tube.
5. Block in Incubation Buffer for 10 minutes on ice.
6. Add the unconjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the dilution recommendations).
7. Incubate for 1 hour on ice.
8. Rinse as before in Incubation Buffer by centrifugation.
9. Resuspend cells in fluorochrome-conjugated secondary antibody* diluted in total volume Incubation Buffer at the recommended dilution.
10. Incubate for 30 minutes on ice.
11. Rinse as before in Incubation Buffer by centrifugation.
12. Resuspend cells in 0.5 ml PBS and analyze 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