Miltenyi isolation kits

B cell proliferation kit (R&D system, Abingdon, UK). BD CellFix (BD Biosciences, Oxford, UK). Native human FV, FVa, Thrombin, Dynabeads Human T-cell activator, and CFSE kits (Thermofisher Scientific, Loughborough, UK). Human Factor V ELISA Kit (ab137976, Abcam, Cambridge, UK). Unless otherwise indicated, all reagents were from Sigma-Aldrich Company Ltd (Dorset, UK).
CD4 T cell, CD8 T cell and B cell were isolated using Miltenyi isolation kits (Miltenyi Biotec, Surrey, UK) following the manufacturer’s instruction. Cells were stained with CFSE using CFSE kits (Thermofisher Scientific, Loughborough, UK) following the manufacturer’s instruction. 5 × 10⁴ labelled T cons were mixed with Dynabeads Human T cell activator CD3/CD28 (Thermofisher Scientific, Loughborough, UK) at cell-to-beads ratio of 2 to 1 and incubated at 37°C. Cells incubated with native human FV, FVa, FV constructs at the concentrations indicated, thrombin (5 μg/mL), and hirudin (5 iU/ml) were collected and analysed using a BD Fortessa flow cytometer after 4 or 5 days.

Naïve CD4⁺ or CD8⁺ T cells were isolated from the spleen and lymph nodes of female Balb/c mice using Miltenyi isolation kits (Miltenyi Biotech, Paris, France). Naive CD4⁺ T cells (CD62L⁺) were differentiated into Th1 or T_reg cell subtypes using cytokines and blocking antibody cocktails: anti-IL-4 (10 µg/ml) and IL-2 (10 µg/ml) for Th1 and anti-IFN-γ (10 µg/ml), anti-IL-4 (10 µg/ml), and TGFβ (4 ng/ml) for T_reg cells (24 (link)). During T-cell differentiation, Th1 and T_reg cells were treated with 1 µM of H89 for 72 h and T-cell differentiation was assessed by RT-qPCR. Total CD4⁺ T cells (CD62L⁻) were obtained using the negative fraction and were treated with 1 µM of H89 for 2 h to assess intracellular potassium [K⁺]_i concentration, as previously reported (25 (link)). CD8⁺ T cells were cultured for 72 h with 1 µM of H89 to assess the level of Akt phosphorylation and granzyme B production. All T cells were activated using anti-CD3/anti-CD28 Dynabeads (Gibco - Thermo Fischer Scientific, Waltham, MA, USA) and cultured in antibiotics-enriched RPMI medium [10% FBS with 1% penicillin, streptomycin, and amphotericin B (PSA)].