Okt3 86022706

Primary human CD3⁺ CD4⁺ CD27⁺ CD28⁺ T cells (5 x 10⁶) were transfected with 3mg of TERT KO CRISPR/Cas9 plasmid (h) (sc-400316, Santa Cruz Biotechnology) according to the manufacturer instructions. TERT-KO (GFP⁺) T cells were then activated by anti-CD3 (0.5 μg/ml; OKT3 86022706 Sigma Aldrich used throughout) plus anti-CD28 (0.5 μg/mL; 37407 MAB342 R&D systems used throughout) and then purified by FACS 96h post-transfection. Control primary human CD3⁺ CD4⁺ CD27⁺ CD28⁺ T cells were transfected with 3mg of control CRISPR/Cas9 Plasmid (sc-418922, Santa Cruz Biotechnology). Knock-out efficiency was confirmed by qPCR (Hs00972656_m1; TaqMan). Transcript expression was normalized to that of the housekeeping gene GAPDH by the change-in-cycling-threshold method (ΔΔCt) for the calculation of values supplied by Applied Biosystems. In experiments with telomerase enhancement, 10⁷ primary human CD3⁺ CD4⁺ CD27⁺ CD28⁺ T cells were transfected with 3 μg of CRISPR telomerase activation particles (sc-400316-ACT, Santa Cruz Biotechnology) and then used 96h post transfection. For immunoblots to TERT (1:1000; sc-7212, Santa Cruz Biotechnology) on purified GFP⁺ primary human T cells was used as previously described¹. Control was control CRISPR/Cas9 Plasmid as advised by the manufacturer (sc-437275, Santa Cruz Biotechnology).