Journal: PLOS ONE

Article Title: THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets

doi: 10.1371/journal.pone.0278365

Figure Lengend Snippet: Three different images are shown of IgG-opsonized platelets incubated with THP-1-CD16A macrophages. Platelets were labelled with 5- chloromethylfluorescein diacetate (CMFDA) (green) before phagocytosis. Non-phagocytosed platelets were differentiated after phagocytosis using an AF647-conjugated anti-human CD42a antibody (red). Platelets were additionally defined by size (roughly 1.5 to 3.5 μm) to distinguish them from internalized microparticles or platelet aggregates. THP-1-CD16A macrophages were observed by spinning-disc confocal microscopy under 63x objective oil immersion with differential interference contrast and DAPI stain on a Quorum multi-modal imaging system (Quorum Technologies, Ontario, Canada). Four images were taken at the center of each well with Z-stacking. Phagocytosis was quantified using Imaris v9.6.0. Scale bar = 3 μm. White arrows indicate examples of phagocytosed platelets.

Article Snippet: Alexa Fluor 647-conjugated mouse anti-human CD42a (clone GRP-P) was purchased from BioRad Laboratories (California, USA).

Techniques: Incubation, Confocal Microscopy, Staining, Imaging

Journal: Biological Procedures Online

Article Title: Refining Flow Cytometry-based Sorting of Plasma-derived Extracellular Vesicles

doi: 10.1186/s12575-025-00293-2

Figure Lengend Snippet: Characterization of CD42a+ EVs. a Representative dot plots for EVs stained with 1mM CFDA-SE at 4°C ( a ) and at RT ( b ). Representative contour plots of CFSE+ EVs stained with either APC-conjugated CD42a antibody d or corresponding non-reactive immunoglobulin of the same isotype ( c ). These analyses have been performed using the 70 mm nozzle. e Histograms showing the normalized diameter (nm), as determined using Rosetta Calibration beads, for pre-sorted CD42a+ events. f Histogram representing the relative expression levels of 16 miRNAs analyzed on both CD42a+ (orange bars) sorted EVs and platelets (blue bars) derived from the same donors

Article Snippet: The expression of the markers CD9 (APC Mouse Anti-Human CD9, Clone HI9a, 312108; BioLegend), CD63 (PE-Cy7 Mouse Anti-Human CD63, Clone H5C6, 561982; BD Biosciences) and CD42a (CD42a antibody, anti-human, APC, REAfinity TM, Clone: REA209, 130-123-831; Miltenyi Biotec) was evaluated within the CFDA-SE-positive events and compared to the corresponding isotype controls.

Techniques: Staining, Expressing, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: T granules in human platelets function in TLR9 organization and signaling

doi: 10.1083/jcb.201111136

Figure Lengend Snippet: TLR9 does not colocalize with known PLT α granule, dense granule, lysosomal, or endosomal markers. Washed human whole-blood PLTs were spun down onto poly- l -lysine–coated glass cover slides, permeabilized with 0.5% Triton X-100 for 5 min, and probed for TLR9. (A) PLTs were colabeled for α granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, and CD42b), dense granule (serotonin), lysosome (LAMP-1), or endosome (M6P and syntaxin-13) protein markers using two separate colors. Insets represent magnified regions outlined by the yellow boxes for each image. (B) TLR9 labeling of whole-blood PLTs from patients with Gray PLT and Hermansky–Pudlack syndromes was compared with human normal PLT controls. All samples were examined by wide-field fluorescence microscopy. (C) 2D intensity scatter plot analysis of image overlays reveal that, although TLR9 colocalizes well with PDI, it does not colocalize with either TLR7 or TLR8. These data suggest that TLR9 and PDI may be distributing to a unique intracellular body (T granule) underlying the plasma membrane in resting human PLTs. (insets) Bars, 2 µm.

Article Snippet: For granule localization, samples were incubated with primary antibodies against TLR9 (Imgenex), VEGF, fibrinogen, CD62P, CD42a, PDGF-B (Santa Cruz Biotechnology, Inc.), CD42b (Dako), M6P, LAMP-1 (Abcam), Syntaxin-13 (a gift from A. Peden, University of Cambridge, Cambridge, England, UK), serotonin (EMD Millipore), or VAMPs 5/7/8 (Novus Biologicals) alone or in combination.

Techniques: Labeling, Fluorescence, Microscopy, Clinical Proteomics, Membrane