Specificity of antibodies under “mild” permeabilization conditions andcell-surface expression of endogenous CCR5 after adhesion of THP-1 cells. (A-C) Specificity of antibodies used in Achour et al²  under “mild” permeabilization conditions. (A) CHO-K1 cells transfected with CCR5-YFP or a control pcDNA3 plasmid were fixed with 4% paraformaldehyde (PFA) for 20 minutes at 4°C. After quenching with 50mM NH₄Cl in PBS for 15 minutes, cells were washed with phosphate-buffered saline (PBS) and then incubated with PBS-bovine serum albumin (BSA) 1% containing 0.1% saponin for 20 minutes. After 1 hour incubation with the 2D7 anti-CCR5 antibody they were washed several times and incubated with Cy3-conjugated anti-mouse IgG in the same buffer. After extensive washing, slides were mounted in DAPI-containing medium. Confocal microscopy images (obtained with a Leica spinning-disk microscope equipped with a CoolSnap HQ2 CCD camera) were processed with ImageJ 1.43u software. Scale bar. 6 μm. (B) THP-1 cells analyzed for the expression of endogenous CCR5 as in panel A; saponin was omitted in nonpermeabilized cells (NP); (P):permeabilized. Control CCR5-negative Jurkat T cells are shown for comparison (C) FACS experiments in permeabilized CHO-K1 and THP-1 cells. Top panels: CHO-K1 cells were transfected with CCR5-YFP (left) or control YFP (right) plasmids. After fixation in 2% PFA for 20 minutes, cells were washed with PBS-1% BSA, 10mM HEPES, and 0.5mM EDTA, followed by 2 additional washes in the same buffer containing 0.1% saponin. Cells were then incubated in the same permeabilization buffer with the 1/85a Alexa Fluor 647–conjugated anti-human CCR5 antibody (1:50) for 45 minutes. After 2 washes, 1/85a Alexa Fluor 647 antibody signals gated on YFP-positive (blue histograms) or -negative (red histograms) cell populations were analyzed. Bottom: panels: after fixation, THP-1 cells were washed 2 times with PBS-0.2% BSA, 10mM HEPES, 0.5mM EDTA containing (permeabilized) or not (nonpermeabilized) 0.1% saponin. Cells were then incubated for 45 minutes in 100 μL of the appropriate buffer, containing (or not) 0.1% saponin and the 1/85a Alexa Fluor 647–conjugated anti-human CCR5 antibody (1:50) or the isotypic control (same antibodies as in Achour et al² ). 1/85a Alexa Fluor 647 antibody signal (green histograms) or isotypic control signal (gray histograms) are shown. The percentage of positive cells is also indicated. FACS experiments were also conducted with the 2D7 antibody and gave similar results (not shown). (D-F) Increasing cell-surface expression of endogenous CCR5 after adhesion of monocytic THP-1 cells. (D) THP-1 cells were left untreated (left panels) or were incubated on fibronectin-coated (5 μg/mL Superfibronectin, S5171, Sigma-Aldrich) glass coverslips for 10 minutes (right panels), fixed (without permeabilization) and stained for surface CCR5 (top panels) or CD4 (bottom panels), using mouse 2D7 anti–human CCR5 or mouse OKT4 anti–human CD4, respectively. After incubation with the anti-mouse Alexa Fluor 488–conjugated antibody, cells were examined with a confocal microscope. Scale bar, 10 μm. (E) Average values of CCR5 surface expression calculated in 40 cells analyzed in panel D. NA indicates nonadherent; A, adherent. Fluorescence quantification (in arbitrary units [AUs[) was performed using ImageJ Version 1.38X software, with background subtraction. Statistical significance (**P < .01) using Student t test. BFA: cells incubated with 2 μg/mL brefeldin A (Sigma-Aldrich) overnight before the experiment. (F) Quantization of CD4 surface expression as described in panel E.