Human trustrain fcx

The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10⁵ per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4°C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4°C for 30 min. Cells were then washed with PBS and resuspended in 300 μL PBS for study.

Flow cytometry was performed with a MACSQuant 10 flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data was analyzed using the FlowJo software version 10.0 (FlowJo, LLC, Ashland, OR, USA). Antibody titrations and compensation was performed in beforehand. To measure T-cell activation, PD1/PD-L1 expression, and T-cell platelet aggregate formation, the following antibodies were used: CD4-PE-Cy7, CD8-BV-510, PD1-PE, PD-L1-APC, CD41-FITC (all from Biolegend, San Diego, CA, USA). T-cell Proliferation was assessed by the decreasing fluorescence intensity of the proliferation-dye. Human TruStrain FcX (BioLegend) was added before staining to avoid unspecific binding of the antibodies. Gating strategy of CD4 and CD8 positive T lymphocytes, as well as PD-1 and PD-L1 positives and proliferated cells, are shown in Figure 1.