Percp cy5.5 cd45ra

Flow cytometry was used to measure the expression of cell markers (LSR II, BD Biosciences). Quantification of α4β7 levels was performed by gating β7⁺CD45RA⁻ within the population of CD4⁺ T cells to identify α4β7^hi populations based on previously published data that demonstrated that αEβ7, the only other form of β7 found on human T cells, is rarely found in the blood [2 (link),13 16 (link),17 ]. PBMCs were stained with the following combination of antibodies from BD Biosciences: APC-H7-CD3 (560176), PerCP-Cy5.5-CD45RA (563429), BV421-CCR5 (562576), PE-Integrin β7 (555945), BB515-CD25 (564467), PE-Cy7-CD27 (560609), PE-CF594-HLA-DR (562304), BUV395-CD8 (563795), BUV496-CD38 (564657); and the following antibodies from Biolegend: BV605-CCR6 (353420), AlexaFluor 700-CD4 (344622), BV510-CCR7 (353232), AlexaFluor 647-CD127 (351318) (Supplemental Figure S1).

The 2 × 10⁵ tumor cells or PBMCs were resuspended in 100 μL of culture medium and were incubated with fluorescent reagents for 30 min at room temperature. To detect T cell markers in PBMCs, cells were incubated with fluorescent-conjugated antibodies including APC/Cy7-CD3, PE/Cy7-CD4, Alexa488-CD8, PE-Cy5.5-CD8, PE-CD16, PerCP/Cy5.5-CD25 (IL2Rα), PerCP/Cy5.5-CD28, Pacific blue-CD45, PerCP-Cy5.5-CD45RA, APC-CD122 (IL2Rβ), PE-CD178 (FasL), Alexa700-CD183 (CXCR3), FITC-CD197 (CCR7) and APC-CD279 (PD-1) (BioLegend, San Diego, CA, USA). To detect cell apoptosis in A549 cells treated with 20 Gy of irradiation, cells were incubated with Annexin V-FITC and Propidium Iodide (Strong Biotech Corporation, Taiwan). Cells were consequently added to 900 μL of PBS buffer containing 0.1% of FBS and analyzed using an Attune NxT Flow Cytometer (Invitrogen, Waltham, MA, USA).