5 × 104 UniCAR T cells were incubated alone or in the presence of 1 × 104 tumor cells in a 96-well U-bottom plate. Recombinant Abs were added at a concentration of 5 nM. After 24 h, cell-free supernatant was harvested and analyzed for secreted cytokines by ELISA as previously described [45 (link)]. Human TNF ELISA Set, Human IFN-Gamma ELISA Set, Human IL-2 ELISA Set, and BD OptEIA Reagent Set B were purchased from BD Biosciences (Becton Dickinson GmbH, Heidelberg, Germany) and used according to the manufacturer’s instructions.
After 24 h of co-cultivation, UniCAR T cells were further examined with respect to their activation status. For this purpose, cells of one triplicate were pooled and stained with anti-human CD3-PE-Vio®770 or CD3-VioBlue and anti-human CD69-APC Abs (all Miltenyi Biotec GmbH) for 15 min at 4 °C. To allow exclusion of dead cells during analysis, counterstaining with 1 μg/mL propidium iode/PBS solution was performed. Flow cytometric data were acquired with the MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).
After 24 h of co-cultivation, UniCAR T cells were further examined with respect to their activation status. For this purpose, cells of one triplicate were pooled and stained with anti-human CD3-PE-Vio®770 or CD3-VioBlue and anti-human CD69-APC Abs (all Miltenyi Biotec GmbH) for 15 min at 4 °C. To allow exclusion of dead cells during analysis, counterstaining with 1 μg/mL propidium iode/PBS solution was performed. Flow cytometric data were acquired with the MACSQuant Analyzer 10 (Miltenyi Biotec GmbH).
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