Pe anti human cd45ro

The following cell surface fluorochrome-conjugated monoclonal antibodies were used to detect T cells phenotype: FITC anti-human CD4, PE/Cy7 anti-human CD8, PE anti-human CD3 (Biolegend, San Diego, CA, USA) and APC anti-human NKG2D (eBioscience). T cell activity was determined by staining the surface APC anti-human CD69, intracellular PE anti-human IFN-γ (BD Biosciences, San Diego, CA, USA), PE anti-human GzmB (eBioscience, San Diego, CA, USA) and PE anti-human Bcl-2 (Biolegend) antibodies. PE anti-human PD-1 and APC anti-human Tim-3 were purchased from Biolegend. FITC anti-human CD45RA, Percp-cy5.5 anti-human CCR7 and PE anti-human CD45RO were purchased from Biolegend to determine T cell subsets. APC Annexin-V and 7-Aminoactinomycin D (7-ADD) from BD Biosciences were used for apoptosis staining. APC mouse IgG1, κ isotype, PE mouse IgG1 isotype, PE/Cy7 mouse IgG2a, κ isotype, FITC mouse IgG1 isotype, PE/Cy7 mouse IgG1, κ isotype (Biolegend) were used as controls. Flow cytometry was performed on the BD LSRFortessa flow cytometer, and data were analyzed using FlowJo Version 10 software.

We extracted peripheral blood mononuclear cells (PBMCs) by utilizing lymphoprep gradient density centrifugation (AS1114544, Axis-Shield, Norway). PE anti-human CD45RO (304206, Biolegend, USA), APC anti-human CD45RA (304112, Biolegend), FITC anti-human CD8 (100706, Biolegend), PE-cy5 anti-human CD4 (CD45-T2-100, Alpha Diagnostic International), Alexa Fluor^® 488 anti-human CD25(356115, Biolegend) and PerCP anti-human CD3(344813, Biolegend) were used to stain PBMCs, which are co-cultured with OSRC-2 for 48 hours. CD45RO+CD8+ T lymphocytes and CD45RA+CD8+ T lymphocytes were extracted by BD FACSAria II (Becton Dickinson, USA). The fineness of all extracted PBMCs conventionally surpassed 90%. We assembled and suspended 1×10⁶ cells in phosphate-buffered saline (PBS) and then cultured for 5 min at 4°C. Subsequently, the isolated cells were cultured on crushed ice for 0.5 hour in 50 μl of staining solution with 1 μg/ml of corresponding conjugated antibodies. The stained cells were rinsed twice with the pre-cooling PBS. All the samples were examined utilizing the FACS Calibur flow cytometer (BD) and data were analyzed utilizing CellQuest software (BD).