Cd45 pe clone 30 f11

Dissociated retinal cells from AAV8(Y733F).CAG.GFP.WPRE-injected mice were stained with the viability dye ViaKrome 405 (Beckman Coulter) at 1/1,000 dilution in PBS for 20 min on ice. Cells were subsequently incubated with TruStain FcX PLUS Fc block (2.5 μg/mL) (BioLegend, London, UK) on ice for 10 min. Cells were washed with staining buffer prior to cell surface antigen staining. Retinal cells from AAV8.CAG.GFP.WPRE- and AAV8.CAG.hREP1.WPRE-injected mice were incubated in Fc block immediately after dissociation and were subsequently stained with cell surface markers. Antibody staining was performed in all retinae on ice for 20 min, with samples protected from light. Cells were subsequently washed with staining buffer prior to flow cytometric analysis. Retinal cells from AAV8.CAG.GFP.WPRE- and AAV8.CAG.hREP1.WPRE-injected mice were stained with 4′,6-diamidino-2-phenylindole (DAPI) (BioLegend, London, UK) 5 min prior to flow cytometric analysis.
The antibodies used in this study were as follows: CD45 Brilliant Violet 711 clone 30-F11 (0.5 μg/mL), CD45 PE clone 30-F11 (0.5 μg/mL), CD11b-APC clone M1/70 (0.25 μg/mL), CD3-Alexa Fluor 700 clone 17A2 (5 μg/mL), CD4 Brilliant Violet 785 clone RM4-5 (0.5 μg/mL), CD19 APC/Fire 750 clone 6D5 (2 μg/mL), NK-1.1 PE/Cyanine7 clone PK136 (1 μg/mL), and CD11c PerCP/Cyanine5.5 clone N418 (1 μg/mL) (all from BioLegend, London, UK).

Blood from healthy mice was drawn via cardiac puncture with a heparinised syringe. The blood was diluted 1:2 with PBS and immediately stained with fluorescent antibodies (CD41-BV421, clone MWReg30, BioLegend; CD45-PE, clone 30-F11, BioLegend; Ly-6B.2-FITC, clone 7/4, Bio-Rad) in 1:200 dilution. Samples were stimulated simultaneously with 1 µM ADP (Sigma-Aldrich) where indicated. After 20 min, the reaction was stopped by fixing the samples in 2% PFA (Roth) and 15 min later diluted again 1:10 in PBS and subsequently analysed via imaging flow cytometry using the AMNIS^® ImageStream^® II (Luminex Corporation, Austin, TX, USA).

Platelets and bone marrow neutrophils were isolated as described above. Platelets and neutrophils were combined at a 10:1 ratio in a final volume of 100 µL RPMI (PAN) containing 0.5% FCS (PAN) and 25 mM HEPES (Sigma). The cells were stimulated with 10 µM fMLP (Sigma) and 0.1 U/mL thrombin (Sigma) for 10 min at RT with subsequent antibody staining (CD41-BV421 (clone MWReg30, BioLegend), Gr-1-AF633 (clone RB6-8C5, purified from hybridoma supernatant), CD45-PE (clone 30-F11, BioLegend), Ly6B.2-FITC (clone 7/4, Bio-Rad)). CD41⁺ signals were compared to isotype controls (BV421 Rat IgG1, κ isotype ctrl antibody, BioLegend) and measurements were performed with a FACSCantoII flow cytometer.