Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

Intracellular p24 Assay by Flow Cytometry (Caltag)


  • Coulter anti-p24 antibody KC57-FITC ( 6604665)*. Store at 4°C.

  • Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control ( 20074A). Store at 4°C.

  • Caltag Laboratories Fix Perm permeabilization kit ( GAS-004).

  • Falcon 5 ml FACS-tubes ( 2054).

  • PBS/1%BSA: dissolve 4 mg Sigma BSA ( A-2153) in 400 ml PBS. Filter through 0.22µm filter. Store at 4°C.

  • 2X Storage buffer (2% Formaldehyde, 0.05% Glutaraldehyde): add 5.71 ml 35% Formaldehyde and 200 µl 25% Glutaraldehyde to 94ml PBS. Store at 4°C.


  1. For each sample to be analyzed use up to 0.5 x 106 cells (see note).

  2. Pellet cells in 5 ml FACS-tube (1100 rpm for 5 minutes).

  3. Resuspend in 100 µl of reagent A (Fixation Medium).

  4. Incubate for 15 minutes at room temperature.

  5. Add 5 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

  6. Remove supernatant and resuspend cell pellet in 100 µl of reagent B (Permeabilization Medium) and 1 µl of the appropriate antibody (anti-p24 or isotype control).

  7. Vortex at low speed for 1-2 seconds.

  8. Incubate for 15 minutes at room temperature (in the dark).

  9. Wash cells with PBS/1% BSA as described above.

  10. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml of 1.0% Formaldehyde (1x Storage Buffer) and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours.

Note: for each cell sample that is being tested for p24-expression, be sure to also test control (i.e. test 0.5 x 106 cells with p24-specific antibody and 0.5 x 106 cells with the isotype control antibody) for aspecific sticking.

*Also available: anti-p24 antibody KC57-RD1 ( 6604668) for use on GFP-expressing cells. Isotype control: Pharmingen PE-conjugated mouse IgG2a, kappa isotype control ( 20075A).