Igg1κ pe

All samples were collected under a protocol approved by the Ethics Committee of China Pharmaceutical University, following written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from an anonymous healthy donor’s blood by Lymphoprep™ reagent (Axis-Shield). Isolated CD4⁺ T or CD8⁺ T cells were immediately enriched by positive selection with magnetic microbeads (Miltenyi Biotec). The two isolated T cell subtypes were cultured in human T cell complete medium, consisting of X-VIVO15 (Lonza), 10% (v/v) fetal bovine serum (FBS, Biological Industries), and 10 mM N-acetyl L-Cysteine (Sigma-Aldrich), supplemented with 40 and 80 IU/mL of recombinant human IL-2 (PeproTech) for CD4⁺ T and CD8⁺ T cells, respectively. The sorted T cells were resuspended in phosphate buffer saline (PBS) and stained with an anti-CD4-PE (phycoerythrin) antibody (BD Pharmingen) or an anti-CD8-APC (allophycocyanin) antibody (BD Pharmingen). The control groups were designed and stained with an isotype antibody IgG1κ-PE (BD Pharmingen) or IgG1κ-APC (BD Pharmingen), respectively. All stained cells were washed three times in PBS and processed with a flow cytometer.

The antibodies employed were anti-TLR4-PE antibody (BD Biosciences, Franklin Lakes, NJ, USA), a monoclonal antibody targeting TLR4 expressed on the platelet cell surface; and anti-CD41a-FITC (BD Biosciences), which is a platelet-specific monoclonal antibody that, without needing to be activated, recognizes the platelet GPIIb/IIIa complex. Nonspecific binding was investigated using IgG1 κ-FITC and IgG1 κ-PE antibodies (BD Biosciences). Platelets were stimulated using thrombin (Sigma, St. Louis, MO, USA). In our laboratory, we prepared the pH 7.4 platelet wash buffer (20 mM HEPES, 145 mM NaCl, and 9 mM Na₂EDTA) and pH 7.4 HEPES-buffered Tyrode’s solution (10 mM HEPES, 1.61 mM KCl, 0.42 mM Na₂HPO₄, 11.9 mM NaHCO₃, 136.89 mM NaCl, 1.05 mM MgCl₂, and 5.6 mM glucose).

Cells were harvested by trypsinisation, counted using the trypan blue exclusion method, and 1 × 10⁶ cells were transferred to 12 × 75 mm polycarbonate tubes. The cells were then washed twice in 2 mL flow cytometry buffer (0.5% (w/v) BSA in DPBS) by centrifugation at 400 g for three min at 4 °C. Cells were then fixed in 2% (w/v) paraformaldehyde for 10 min at 37 °C, washed, and then permeabilised with 1 mL 90% (v/v) ice-cold methanol on ice for 30 min. Following permeabilisation, the cells were washed and then incubated with a PE-conjugated murine anti-vimentin mAb (clone RV202, BD Biosciences) or the corresponding PE-conjugated mouse isotype control (IgG1κ-PE, BD Biosciences), in accordance with the manufacturer’s instructions. CD44 and CD24 staining was achieved by staining the cells without permeabilisation using FITC conjugated antiCD44 (eBioscience, clone 24E10) and APC conjugated CD24 (eBioscience, clone eBioSN3) antibodies. Prior to flow cytometric analysis, the cells were washed twice and then re-suspended in 300–400 μL of Isoton solution (Beckman Coulter) on ice. Cells were analysed using a Beckman Coulter Gallios flow cytometer. Histograms and dot plots were derived and analysed using Beckman Coulter Kaluza version 1 software.