Reagents

  • Pharmingen anti-CCR5 antibody 2D7-FITC (cat.no. 36464X)*. Store at 4°C. Use at 1:5 dilution.

  • Pharmingen anti-CXCR4 antibody 12G5-PE (cat.no. 36195X). Store at 4°C. Use at 1:100 dilution.

  • Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control (cat.no. 20074A). Store at 4°C. Use at 1:100 dilution.

  • Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A). Store at 4°C. Use at 1:100 dilution.

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

  • PBS/1% BSA: dissolve 4mg Sigma BSA (cat.no. A-2153) in 400ml 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 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 5 min).

  3. Resuspend in 100 µl of cold PBS/1%BSA with appropriate concentration of specific antibody or isotype control.

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

  5. Incubate for 30 minutes in the dark at 4°C.

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

  7. 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 chemokine receptor-expression, be sure to also test control (i.e. test 0.5 x 106 cells with chemokine receptor-specific antibody and 0.5 x 106 cells with the isotype control antibody) for aspecific sticking.

*Also available: anti-CCR5 antibody 2D7-PE (cat.no. 36465X) 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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