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Flow Cytometry Protocol for Intracellular 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 1XPBS. 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 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 hemacytometer 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.