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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Nat Protoc. 2020 Feb 26;15(4):1507–1524. doi: 10.1038/s41596-020-0294-8

Fig. 3. Steps in the generation of CAR+ primary human T cells from human whole blood.

Fig. 3

a, Cells isolated from human donor whole blood using a CD8+ or CD4+ enrichment kit, as in steps 1–7, were stained with antibodies for CD3 and CD8 (left) or CD3 and CD4 (right). b, CD4+ T cells were transduced with lentivirus encoding a TGF-β CAR with a short extracellular spacer domain, a TGF-β CAR with a long extracellular spacer domain, or an scFv-less CAR (as in steps 10–33). Each CAR was linked via a self-cleaving peptide to truncated EGFR (EGFRt). On Day 9 of culture, transduction efficiency was evaluated by staining for EGFRt. c, EGFRt-expressing cells were tagged with Erbitux-Biotin, then Anti-Biotin MicroBeads, and sorted through magnetic columns twice (steps 34–42). The sorting process was monitored by staining for both EGFRt and FLAG-tagged CARs in the flow-through and elution fractions.