Apc conjugated anti human cd3 antibody

To detect the expression of CCR5 in cells edited by CRISPR/SaCas9, we collected the control-modified and CCR5-sgRNA/SaCas9 modified TZM-bl cells 3 days post transfection and washed with cold 1 × PBS for three times. Then the cells were strained with APC conjugated anti-human CCR5 antibody (Biolegend) for 30 min on ice and analyzed by flow cytometry (FACS AriaIII, BD). To count the number of CD4⁺ T cells in blood obtained from the mice in different time points, we firstly treated the blood samples by red blood lysis buffer (BD Biosciences) for 15 min, then stained the samples with PE conjugated anti-human CD4 antibody (BD Biosciences), APC conjugated anti-human CD3 antibody (BD Biosciences) and FITC conjugated anti-human CD8 antibody (BD Biosciences) for 30 min on ice. At last, all samples were analyzed by flow cytometry (FACS AriaIII, BD) and Flow Jo software (Treestar). For western blotting assay, we lysed the cells with lysis buffer containing 50 mM Tris-HCl pH = 7.4, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1.5 Mm EDTA, 0.25% deoxycholate and protease inhibitor PMSF and cocktail (Roche Applied Science) on ice for 30 min. The lysates were centrifuged by 12,000 rpm for 10 min and the supernatants were mixed with 2 × SDS loading buffer incubation at 100 °C for 10 min. The proteins were detected by SDS-PAGE with anti-GAPDH (Proteintech) and anti-CCR5 antibodies (Proteintech).

Raji cells (1.6 × 10⁵ /well) were divided into three treatment groups, given CAR-T cells (2 × 10⁴ /well) with or without GA (100 μM), GA (100 μM) and the control (PBS) group (n = 3). After incubation for 12 hours, the cells were stained with APC-conjugated anti-human CD3 antibody (BD Pharmingen, USA) and FITC-conjugated Annexin V (Gene-Protein Link, China) for half an hour. The apoptosis rate of Raji cells was then measured by flow cytometric analysis (CytoFLEX, Beckman Coulter, USA), and FACS data was analyzed by CytoFLEX Software (Beckman Coulter, USA) and FlowJo (Version 10.0.7).

FITC-conjugated human CD19 antigens (Cat. No. CD9-HF2H2; Acro Biosystems, Newark, NJ, USA) were used for determining the expression rate of the VHH-based and scFv-based CAR molecules on the surface of VHH-CAR-Ts and scFv-CAR-Ts, respectively, in the flow cytometry assay. The concentration of the FITC-conjugated CD19 antigen and the number of cells used for the flow cytometry assay were according to the manufacturer’s instructions. APC-conjugated anti-human CD3 antibody (BD Pharmingen™, USA; Cat No. #555335) was used to assess the percentage of CD3-positive cells in a given population of PBMCs, according to the manufacturer’s instructions.