Apc cy7 conjugated anti cd8

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)}.

After transfection (24 h), primary CD8⁺T cells were stimulated using anti-CD3/CD28 (3 μg/ml) for 24 h. The protein transport inhibitor (GolgiStop; 1 μl/ml, BD Biosciences) was added to the culture for the last 6 h. The cells were stained with PE-cy7-conjugated anti-CD3 and APC-cy7-conjugated anti-CD8 (Biolegend). Subsequently, intracellular staining was performed by incubating the cells in 1X Perm/Wash Buffer for 15 min in the dark, followed by incubation with APC-conjugated anti-IFN-γ and FITC-conjugated anti-granzyme B for 30 min at 4°C. After staining, the cells were fixed in 1% formaldehyde. The intracellular expression of IFN-γ and granzyme B was determined using a BD LSR II flow cytometer and data were analyzed using the FlowJo software.

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).