Mullins Molecular Retrovirology Lab

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.

• Caltag Laboratories Fix & Perm permeabilization kit (cat.no. GAS-004).

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

• PBS/1%BSA: dissolve 4 mg Sigma BSA (cat.no. 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.

 

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