Anti 7 aad antibody

The goat-F(ab’)2 anti-human IgG antibody (Southern Biotech, Birmingham, AL, USA) directed against the extracellular IgG1 CH2-CH3 CAR domain was used to determine the CAR expression on T cells. TCR expression on the cell surface of T cells was analyzed using an MHC Dextramer (HLA-A*0201/YLEPGPVTV; Immudex, Copenhagen, Denmark) directed against the gp100-specific TCR. In addition, the anti-7-AAD antibody (BD Biosciences) was used to exclude nonviable T cells. The detailed procedure of cell surface staining was previously described [44 (link)]. Immunofluorescence was measured via the FACS Calibur (BD Biosciences, Heidelberg, Germany), which was equipped with the CellQuest Pro software (BD Biosciences). Data were analyzed using the FCS Express software, version 5 (DeNovo Software, Glendale, CA, USA).

In order to analyze the cellular composition of the cell populations before and after expansion, the cells were stained using an anti-CD3 antibody (Clone: UCHT1; BD Biosciences, Franklin Lakes, NJ, USA) in combination with either an anti-CD56 antibody (Clone: NCAM16.2; BD Biosciences) or an anti-CD8 antibody (Clone: SK1; BD Biosciences). Isotype-stained cells served as controls. Additionally, all the cells were stained with an anti-7-AAD antibody (BD Biosciences) to exclude nonviable cells. Prior to staining, the cells were cryopreserved as previously described [19 (link)]. Immunofluorescence was measured via a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with CellQuest Pro software (BD Biosciences). The data were analyzed using FCS Express, version 5 (DeNovo Software, Glendale, CA, USA).