Anti cd45ro percp cy5

We applied BD FACSCalibur and BD FACSAria II instruments for FCM detection. BD FACSDiva software and FlowJo software were used for analysis. FCM detection was performed according to the schedule shown in Figure 2C. CD3, CD4, CD8, and CAR expression were evaluated on days 6, 10, and 14, and memory T subsets were evaluated on days 10 and 14. We used His-tagged recombinant CEACAM5 (CEA-His; Sino Biological Inc., 11077-H08H-50) and biotin-protein L (GenScript, M00097) for specific detection of CAR expression. The following antibodies were used: anti-CD3-PE-Cy7 (BioLegend, 300420), anti-CD4-BUV395 (BD, 564724), anti-CD8-BV510 (BioLegend, 344732), anti-CD25-BV421 (BioLegend, 302630), anti-CD45RA-BV421 (BioLegend, 304130), anti-CD45RO-PerCP-Cy5.5 (BioLegend, 304222), AF-647-conjugated IgG fraction of mouse monoclonal anti-biotin (Jackson, 200-602-211) and anti-CD197-PE (BioLegend, 353204).

The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65_341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).