Anti human cd3 apc cy7

To evaluate T cell activation, freshly isolated PBMCs (1 × 10⁶ cells/well) were activated with SARS-CoV-2 Omicron BA.1 (S1) peptides at a final concentration of 2 µg/mL in R10 medium at 37 °C with 5% CO₂ for 20 h. After incubation, cells were harvested and stained with the following monoclonal antibodies: anti-human CD3-APC-Cy7; anti-human CD4-V450 (Thermo Fisher Scientific, Waltham, USA); anti-human CD8-FITC; anti-human CD62L-APC (BD Pharmingen, San Diego, CA, USA); anti-human CD45RA-PE-Cy7 for 30 min at 4 °C in the dark. All samples were analysed with a BD FACSLyric Flow Cytometer (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo Software version 10 (Flowjo, LLC, Ashland OR, USA). The absolute numbers of lymphocytes and T lymphocyte subsets were calculated from complete blood count data obtained from the Associated Medical Science (AMS) Wellness Center Khon Kaen University.

CD3/CD28 activated T cells were co-cultured in 96-well plate with and without MSCs unprimed or primed in a ratio 1:1 in triplicate for 48 h. Cells were then collected, washed and phosphoflow staining was done using the Perfix Expose Kit from Beckman Coulter (B26976) per manufacturer's instructions. To be able to gate on CD4+ cells the following surface antibodies were used: Live/dead aqua dead cell stain (Thermofisher), anti-human CD3-APC/Cy7, anti-human CD4-PE. The following antibodies were used for intracellular staining: BD Phosflow™ anti-phospho-S6 ribosomal protein-V450 (S235/236), anti-phospho-MTOR pS2448-PE. The following isotype control antibodies were used: Mouse IgG1a-V450 and Mouse IgG1-PE, all purchased from BD biosciences.