Pe conjugated anti ccr7

CAR-T cell differentiation stages were assessed by flow cytometry. CAR-T cells were harvested and washed twice with 1 ml of phosphate-buffered saline containing 2% foetal bovine serum (FBS; Gibco), followed by incubation with the following antibodies: APC-Cy7-conjugated anti-CD8 (344714, BioLegend, California, USA), Alexa Fluor 700-conjugated anti-CD4 (56004942, eBioscience), PE-conjugated anti-CCR7 (353204, BioLegend), PerCP-Cy5.5-conjugated anti-CD45RA (304122, BioLegend), and PE-Cy7-conjugated anti-CD127 (25-1287-42, eBioscience)^{24 (link)}.

All samples were washed twice in 0.1 mL of phosphate-buffered saline (PBS) containing 2% FBS. The transduction efficiency and CD4/CD8 ratio were determined by labeling CAR T cells with a FITC-labeled human CD19 protein, Fc Tag (CD9-HF251, ARCO, Biosystems, Beijing, China), an APC-conjugated antibody against CD8 (catalog no. 344722, BioLegend, California, USA), and a PE-Cy7-conjugated antibody against CD4 (25-0047-42, eBioscience, San Diego, CA) at 4 °C for 45 min in the dark.
For assessing the CAR T cell phenotype to analyze CAR T cell differentiation stages, CAR T cells were incubated with the following antibodies: APC-Cy7-conjugated anti-CD8 (344714, BioLegend), AF700-conjugated anti-CD4 (56004942, eBioscience), PE-conjugated anti-CCR7 (353204, BioLegend), PerCP-Cy5.5-conjugated anti-CD45RA (304122, BioLegend), and PE-Cy7-conjugated anti-CD127 (25-1287-42, eBioscience).
CD19 expression on Raji cells was detected by staining with an APC-conjugated antibody against CD19 (302212, BioLegend), and monocyte CD14 expression was determined by staining with an Alexa Fluor 700-conjugated antibody against CD14 (325614, BioLegend).
After antibody labeling, samples were washed twice in 0.1 mL of PBS containing 2% FBS before detection using an Attune NxT flow cytometer (Thermo Scientific, USA). Data were analyzed using FlowJo V10 (TreeStar, USA).

Spleen and draining lymph nodes (DLNs) were harvested at 12 days following first viral inoculation of the B16-F10 tumor-bearing mice. Before staining, cells were treated with saturating anti-CD16/CD32 (Biolegend, San Diego, CA) in staining buffer (2% FBS and 0.02% sodium azide in PBS). Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience), percp-CY5.5-conjugated anti-CD4 (BD Biosciences Pharmingen), PE-CY7-conjugated anti-CCR4 (Biolegend), and/or PE-conjugated anti-CCR7 (Biolegend). To determine the percentage of CCR4- or CCR7-expressing CD4⁺ T cell population, splenocytes or lymphocytes were gated by plotting forward vs. side scatter followed by gating on CD4⁺ cells. Gated cells were then analyzed for CCR4⁺ or CCR7⁺ cells. Samples were analyzed using BD Biosciences BD-LSR II Analytic Flow Cytometer and FACSDiva software (BD Biosciences Pharmingen).