Cd8 apc clone bw135 80

Isolated T cells were stained with fluorochrome-labeled mAbs directed against human CD4/FITC (clone VIT4), CD3/Vioblue (clone BW264/56) and CD8/APC (clone BW135/80, all purchased from Miltenyi Biotec). For analysis of T cell activation, cells were stained with anti CD3/Pacific Blue mAb (clone UCHT1, BD Bioscience), CD4/PE-Cy7 mAb (clone SK3, BD Bioscience) and CD25/PE mAb (clone BC96, BD Bioscience). For analysis of CD19 expression, Nalm-6 cells were stained with anti-CD19/PE mAb (clone LT19, Miltenyi Biotec). In order to assess binding of the anti-CD19 TM, Nalm-6 cells were incubated with 20 µg/ml of the recombinant protein, followed by the mAb La5B9 directed against the UniCAR epitope (E5B9) [49 (link)] and a PE-conjugated anti-mouse IgG secondary Ab (Beckmann Coulter, Krefeld, Germany). Samples were analyzed using a MACSQuant^® Analyzer and MACSQuantify^® software (Miltenyi Biotec) [49 (link)]. For estimation of UniCAR cell surface expression, the hinge region of the UniCAR contains the peptide tag E7B6 which, like the UniCAR epitope (E5B9), is taken from the nuclear antigen La/SS-B [48 (link), 50 (link)].

Isolated T cells were stained with fluorochrome-labeled mAbs directed against human CD4/FITC (clone VIT4, MiltenyiBiotec), CD3/Vioblue (MiltenyiBiotec) and CD8/APC (clone BW 135/80, MiltenyiBiotec). For analysis of expression of GD2 on JF cells, the cells were stained using the commercial anti-GD2 mAb (Biolegend, San Diego, USA; Clone 14G2a), detected with Alexa Flour 647-labeled goat anti-mouse IgG (Life technologies, Thermo Fisher Scientific). For detection of CAR surface expression T cells were incubated with the mAb 7B6 and subsequently stained with PE-labeled goat anti-mouse IgG (Beckmann Coulter, Krefeld, Germany). Samples were analyzed using a MACSQuant^® Analyzer and MACSQuantify^® software (Miltenyi Biotec) [56 (link)].