Cd42b pe

EMVs were immunophenotypically defined as CD45−/CD42b−/CD31 + (31). Blood samples were pre-diluted 1:100 in buffered saline solution (PBS) and 50 μL of the diluted solution were used for cell-labeling reaction. Anti-human monoclonal antibodies with suitable volumes were added to the diluted sample: 5 μL of FITC CD45 (BD, clone HI30), 2 μL of CD31 Alexa Fluor 647 (BD, clone WM59) and 5 μL of CD42b PE (BD, clone HIP1). The samples containing monoclonal antibodies were incubated for 30 min protected from light at room temperature, and 2 mL of PBS was added; they were then centrifuged for 5 min at 540 g. The supernatant was discarded and the pellet resuspended with 500 μL PBS and 50 μL counting beads (BD Biosciences, San Jose, California) were added. A total of one million events were acquired using a flow cytometer (FACSCanto II) (BD Biosciences, San Jose, California) and analyzed using by Infinicyt version 1.7 (Cytognos, Salamanca, Spain). A blinded investigator processed all samples. The amount of EMVs was determined from the number and volume of counting beads. Microvesicle sizes were compared to platelet sizes using average fluorescence intensity of forward scatter, showing a mean diameter ≤ 1.0 μm. Figure S5 (supplementary material) describes the gating strategy for CD45−/CD42b−/CD31 + EMVs. EMV was assessed 10 min before and 10 and 70 min post-intervention.

HEL cells were seeded into 6-well flat-bottomed plates at an initial cell density of 4 × 10⁴ cells/well. The cells were treated with 10 μM and 20 μM TMEA for 4,8, and 12 days. The medium was added every two days, and the TMEA containing medium was added to the TMEA group. The cells harvested at different time points were washed once with ice-cold PBS and subsequently incubated with 3 μL of CD41-FITC (BioLegend, United States, FHF0411) and 3 μL CD42b-PE (BD, United States, 555473) in 100 μL PBS in the dark for 30 min. Then the cells were re-suspended in 400 μL of PBS, and submitted to the analysis by a FACSVerse flow cytometer (BD Biosciences, SanJose, CA, United States). CD41 expression was assessed using the fluorescein isothiocyanate (FITC) channel, whileCD42b level was examined using the PE channel. The forward and side scatters were used to eliminate the disturbance from cellular fragments. The quantitation of the test biomarker expressions was based on the percentage of CD41⁺/CD42b⁺ cells.

Isolated platelets were resuspended at a concentration of 10⁶/ml of Tyrode buffer (pH 7.4) with 2 mM CaCl₂ and stimulated with designated concentrations of Aß (1–20 ng/ml) for 60 minutes at 37°C and washed once in Tyrode buffer (pH 7.4). Washed platelets were stained with GPIb (CD42b PE, BD Biosciences) for 30 minutes at room temperature in the dark and analyzed in the FSC vs. SSC plot with FSC (E00) settings in the Log scale for cell volume (FCS Geomena) and E01 for detection of microparticles [61] (link).