Reagents

  • Coulter anti-p24 antibody KC57-FITC (cat.no. 6604665)*. Store at 4°C.

  • Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control (cat.no. 20074A). Store at 4°C.

  • Falcon 5 ml FACS-tubes (cat.no. 2054).

  • BD FACS Lysing Solution (cat.no. 349202)

  • BD FACS Permeabilizing Solution (cat.no. 340973)

  • PBS/1% BSA: dissolve 4 mg Sigma BSA (cat.no. A-2153) in 400 ml PBS. Filter through 0.22 µm 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 94 ml PBS. Store at 4°C.

 

Protocol

  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 1x lysing solution.

  4. Incubate for 15 minutes at room temperature.

  5. Add 4 ml of PBS/1% BSA and spin (1100 rpm 5 min).

  6. Remove supernatant and resuspend cell pellet in 100 µl 1x permeabilizing solution.

  7. Incubate for 15 minutes at room temperature.

  8. Add 4 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

  9. Remove supernatant and resuspend cell pellet in 100 µl and 1 µl of the appropriate antibody (anti-p24 or isotype control).

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

  11. Incubate for 30 minutes at room temperature (in the dark).

  12. Add 4 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

  13. 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 (cat.no. 6604668) for use on GFP-expressing cells. Isotype control: Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A).

Department of Microbiology
School of Medicine
University of Washington
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