High levels of intracellular staining for CCR5 by flow cytometery in fixed, permeabilized T cells and GHOST (3) cells that do not express CCR5. (A) Representative histograms of CCR5 staining on fresh and fixed/permeabilized CD4⁺ and CD8⁺ T cells from whole PBMCs. Fresh or fixed/permeabilized cells were initially gated on forward and side scatter. Cells falling into the lymphocyte gate were further gated on being positive for CD3. CD3⁺ cells were further divided into CD4⁺ and CD8⁺ T cells. Histograms reflect CCR5 staining on CD4⁺ T cells (purple) and CD8⁺ T cells (blue) that were either stained unfixed or fixed and permeabilized before staining. CCR5 staining is shown on the x-axis and count on the y-axis. Histogram gates were set with the appropriate fluorochrome-linked isotype control antibody so that ∼ 2% of the population was positive for isotype. (B) Representative histograms of CCR5 staining on fresh and fixed/permeabilized GHOST (3) parental and GHOST (3) Hi-5 (CCR5-transfected) cells. Cells were initially gated on forward versus side scatter. Histograms reflect CCR5 staining on the total cell populations gated on forward and side scatter (purple). CCR5 staining is shown on the x-axis and count on the y-axis. FSC indicates forward scatter; SSC, side scatter; PE-Cy7, phycoerythrin–indocyanine 7; and APC, allophycocyanin.