Figure 1.
THP-1-CD16A macrophages phagocytosed human platelets opsonized with IgG anti-GPIIb/IIIa antibodies. (A) THP-1-CD16A surface expression of FcγRs was evaluated by flow cytometry using fluorescent antibodies to FcγRI (clone 10.1), FcγRIIA (IV.3), all FcγRII isoforms (FcγRIIA/B/C) (AT10), and FcγRIII (3G8). Histograms from a representative experiment are presented. Red shade histogram = unstained control; blue shade histogram = macrophages stained with fluorescent antibody. y-axis, counts (normalized to mode); x-axis, log10 fluorescence intensity. (B) THP-1-CD16A macrophage phagocytosis of platelets opsonized with anti-GPIIb/IIIa mouse IgG2a antibody clone A2A9/6 or human serum positive for HPA-1a alloantibodies (HPA-1a serum). Phagocytosis was quantified from fluorescent photomicrographs as described in the “Methods.” Phagocytic index: number of phagocytosed platelets per 100 counted macrophages. Statistical significance was calculated by performing unpaired, parametric t tests; ∗∗P < .01; ∗∗∗∗P < .001. Data error: mean ± 1 standard deviation (SD). (C) Example of confocal photomicrographs of THP-1-CD16A macrophages after incubation with human platelets opsonized anti-GPIIb/IIIa antibodies (clone A2A9/6, HPA-1a serum) or NHS. White arrows point toward example macrophages with phagocytosed platelets. Platelets were labeled with the cytoplasmic dye 5-chloromethylfluorescein diacetate (CMFDA) (green, top row). External (nonphagocytosed) platelets were identified after phagocytosis using an AlexaFluor 647 (AF647)-conjugated anti-GPIX antibody (red, middle row). Merged fluorescence and differential interference contrast (DIC) images are shown in the bottom row. Areas of red and green colocalization in the merge appear as yellow-orange (nonphagocytosed) vs green (phagocytosed) platelets. Scale bar, 10 μM. Photomicrographs were taken by spinning-disk confocal microscopy under 63× objective oil immersion (numerical aperture 1.47) with DIC and laser fluorescence (488, 647) channels on a Quorum multimodal imaging system equipped with 50 μm pinhole spinning disk and ORCA-Flash 4.0 V2 PLUS sCMOS camera. At least 4 images at distinct locations were taken near the center of each coverslip with Z-stacking every 0.33 μm. Z-stacked images were 3D reconstructed for analysis using Imaris v8.0.2.

THP-1-CD16A macrophages phagocytosed human platelets opsonized with IgG anti-GPIIb/IIIa antibodies. (A) THP-1-CD16A surface expression of FcγRs was evaluated by flow cytometry using fluorescent antibodies to FcγRI (clone 10.1), FcγRIIA (IV.3), all FcγRII isoforms (FcγRIIA/B/C) (AT10), and FcγRIII (3G8). Histograms from a representative experiment are presented. Red shade histogram = unstained control; blue shade histogram = macrophages stained with fluorescent antibody. y-axis, counts (normalized to mode); x-axis, log10 fluorescence intensity. (B) THP-1-CD16A macrophage phagocytosis of platelets opsonized with anti-GPIIb/IIIa mouse IgG2a antibody clone A2A9/6 or human serum positive for HPA-1a alloantibodies (HPA-1a serum). Phagocytosis was quantified from fluorescent photomicrographs as described in the “Methods.” Phagocytic index: number of phagocytosed platelets per 100 counted macrophages. Statistical significance was calculated by performing unpaired, parametric t tests; ∗∗P < .01; ∗∗∗∗P < .001. Data error: mean ± 1 standard deviation (SD). (C) Example of confocal photomicrographs of THP-1-CD16A macrophages after incubation with human platelets opsonized anti-GPIIb/IIIa antibodies (clone A2A9/6, HPA-1a serum) or NHS. White arrows point toward example macrophages with phagocytosed platelets. Platelets were labeled with the cytoplasmic dye 5-chloromethylfluorescein diacetate (CMFDA) (green, top row). External (nonphagocytosed) platelets were identified after phagocytosis using an AlexaFluor 647 (AF647)-conjugated anti-GPIX antibody (red, middle row). Merged fluorescence and differential interference contrast (DIC) images are shown in the bottom row. Areas of red and green colocalization in the merge appear as yellow-orange (nonphagocytosed) vs green (phagocytosed) platelets. Scale bar, 10 μM. Photomicrographs were taken by spinning-disk confocal microscopy under 63× objective oil immersion (numerical aperture 1.47) with DIC and laser fluorescence (488, 647) channels on a Quorum multimodal imaging system equipped with 50 μm pinhole spinning disk and ORCA-Flash 4.0 V2 PLUS sCMOS camera. At least 4 images at distinct locations were taken near the center of each coverslip with Z-stacking every 0.33 μm. Z-stacked images were 3D reconstructed for analysis using Imaris v8.0.2.

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