Flow Cytometry Protocol for Intracellular Staining Using Conjugated Primary 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. Chill tubes on ice for 1 minute.

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 Staining Using Conjugated Primary Antibodies

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

1. Aliquot 5x10⁵ cells into each assay tube (by volume).
2. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation.
3. Resuspend cells in 90 μl Incubation Buffer per assay tube.
4. Block in Incubation Buffer for 10 minutes at room temperature.
5. Add 10 μl of conjugated antibody to the assay tubes.
6. Incubate for 30–60 minutes, in the dark at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

posted June 2005 • revised December 2007