Cd41 clone mwreg30

Platelet rich plasma (PRP) was obtained by centrifuging whole blood at 0.3 G for 6 minutes at 4°C. Platelet concentrations of the PRP were quantified by a cell counter (Beckman Coulter, Brea, CA). PRP was aliquoted in 5 μL samples. Subsequently, 1 μL of CD41 (clone MWReg30, BD Biosciences, San Jose, CA) and CD62P antibodies (clone RB40.34, BD Biosciences, San Jose, CA) were added and allowed to incubate for 20 minutes. Following incubation, 500 μL of phosphate-buffered saline was added. All samples were transferred to flow tubbettes and read on the Attune Flow Cytometer (Life Technologies, Foster City, CA). Activated platelets were identified as CD41(+)/CD62P(+). Platelet counts for each sample were obtained at each timepoint utilizing a Coulter AcT 10 Hematology Analyzer (Beckman Coulter, Brea, California).

To reveal platelet and PCEC activation, deposition, and proliferation, total lung cells were isolated from perfused mouse lung tissues after dissociation with medium containing collagenase/dispase/DNase^{1 (link)}. Retrieved lung cells were incubated with conjugated antibodies and analyzed by flow cytometry on LSRII-SORP (BD)^{51 (link)}. Data were processed with FACSDiva 6.1 software (BD). All doublets were ruled out by FSC-W × FSC-H and SSC-W × SSC-H analysis. Monoclonal antibodies were conjugated to Alexa Fluor dyes or Qdots per manufacturer's protocols (Molecular Probes/Invitrogen), including VE-cadherin (clone BV13, ImClone, 5 μg/ml); CD41 (clone MWReg30, BD Bioscience, 5 μg/ml), and P-selectin (clone RB40.34, BD Bioscience, 5 μg/ml). Single-stained channels were used for compensation. Flow cytometry analysis was performed using various controls, including unstained cells, isotype antibodies, and fluorophore minus one controls for determining gates and compensations in flow cytometry.