Anti cd8 pe antibody

Cell surface CAR expression was assessed using flow cytometry by labelling with anti-MYC antibody (Clone 9B11, Cell Signaling, Danvers, MA, USA) at 4 °C for 45 min and detection of intracellular mCherry expression. Confirmation of the peptide-MHC complex on the cell surface of target cells were performed with staining on ice for one hour, with a recombinant scFv tetramer-Atto-488.
For analysis of T cell activation, CAR T cells and tumour cells were co-incubated for 10 h in T cell media at a 1:1 ratio at 37 °C and 5% CO₂, in triplicate. Positive control for CAR stimulation was provided using plate bound unconjugated MYC-tag (Clone 9B11, Cell Signaling, Danvers, MA, USA), pre-coated overnight 4 °C at in PBS. Cells were then incubated with anti-CD25 APC (Clone PC61, BioLegend, San Diego, CA, USA), anti-CD69 PerCP (Clone H1.2F3, BD Biosciences, East Rutherford, NJ, USA), anti-CD4 FITC (Clone GK1.5, produced at WEHI Bundoora), and anti-CD8 PE antibodies (Clone 53-6.7, BD Pharmingen, East Rutherford, NJ, USA) at 4 °C for 30 min. Cells were analysed on a Fortessa X20 (BD Biosciences, East Rutherford, NJ, USA) and analysis performed on FlowJo software (TreeStar, Ashland, OR, USA). For analysis, cells were gated on live cells, CD4 and CD8, then CD25 and CD69.

We used a FACSCalibur instrument (NovoCyte, ACEA Biosciences, USA) and NovoExpress software (ACEA) for all flow‐cytometric analyses, analyzing at least 20 000 events and isotype antibodies were used as negative controls in all cases. Cells were gently washed twice with cold PBS prior to addition of antibodies. After 30 to 40 min of incubation at 4°C in the dark with fluorescent antibody, the cells were washed twice and resuspended with PBS prior to analysis. T cells phenotypes were analyzed with anti‐CD3 FITC, anti‐CD4 APC, and anti‐CD8 PE antibodies (BD Pharmingen, USA). The 5T4 antigen expression of tumor cells and transfection efficiency of CAR were detected using a FITC‐conjugated goat anti‐human antibody (ZSBIO, China).

Samples were incubated with antibodies diluted in PBS containing 0.2% FCS (FACS buffer) for 30 min at 4°C before samples were analyzed using a Fortessa X20 (BD Biosciences) and analysis was performed using FlowJo software (Becton Dickinson). Gating strategy is shown in Supplementary Figure 1. Antibodies used include anti-MYCTag-AF488 (Clone 9B11, Cell Signaling Technology), anti-CD25-APC (Clone PC61, BioLegend), anti-CD69-BV650 (Clone H1.2F3, BioLegend), anti-CD4-FITC (Clone GK1.5, produced at WEHI Bundoora), and anti-CD8-PE antibodies (Clone 53-6.7, BD Pharmingen).