Flow Cytometry Protocol for Extracellular Staining Using Conjugated Secondary Antibodies

A. Solutions and Reagents

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 distilled water (dH₂O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. 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. Add 5 ml PBS and rinse by centrifugation.
5. Resuspend cells in 5 ml PBS.
6. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Allow 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 primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
6. Incubate for 30-60 minutes at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. Resuspend cells in fluorochrome-conjugated secondary antibody*, diluted in Incubation Buffer according to the manufacturer’s recommendations.
9. Incubate for 30 minutes at room temperature.
10. Rinse as before in Incubation Buffer by centrifugation.
11. 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