Mouse il 2 elisa

For Luminex assays, transduced CAR T cells were incubated in round-bottom 96-well plates (50,000 cells per well) in triplicates for four days in the presence of cytokines after which supernatants were analysed with a Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex Panel (Thermo Fisher Scientific). A cytokine bead array (BD Biosciences) was used to individually measure IFNγ from supernatant of B16-F10 coculture with pmel T cells according to the manufacturer’s instructions. oIL-2 expression from PDA7940b cells (10,000 cells per well, 96-well plate) was evaluated by mouse IL-2 ELISA (Abcam) in cell culture supernatants at various time points after infection with Ad-Null or Ad-oIL-2 (100 VP per cell). In vivo expression was assessed by injecting PBS, Ad-Null or Ad-oIL-2 (1 × 10⁹ VP per tumour) into PDA7940b tumours and collecting 72 h later. Tumours were dissociated by three freeze-thaw cycles and homogenates were analysed for mouse IL-2 by ELISA. Terminal blood was collected by cardiac puncture and processed to serum by centrifugation. IL-2 concentrations were normalized to total protein content.