Cd45ro antibody

One million CAR T cells were stained with primary antibodies for antigen–antibody binding reaction. After 30 min of incubation in the dark, unbound antibodies and solutes were washed with a PBS rinse. This method was used to detect T cell transfection rates, with the following primary antibodies used: CD4 antibody (BioLegend Cat# 357408, USA), CD8 antibody (BioLegend Cat# 344714), CD45RO antibody (BioLegend Cat# 304206), CCR7 antibody (BioLegend Cat# 353214). Target cells were harvested and resuspended in cold PBS containing 2% FBS. The anti-MUC16 antibody was added to the cell suspension for incubation for 1 h. The cells were centrifuged, resuspended, and incubated with Cy3-labeled secondary antibodies in PBS containing 2% FBS for 30 min prior to two washes with PBS. For all flow cytometry experiments, appropriate IgG isotype controls were used to assess nonspecific staining. The specificity of MUC16 antibodies was confirmed by utilizing control IgG matched to the species. All flow cytometry data were collected using a BD LSRFortessa system (BD Biosciences, USA) and analyzed with FlowJo software (FlowJo, Oregon, USA). All flow antibodies are listed in Additional file 8: Table S3.