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. Author manuscript; available in PMC: 2019 Jul 18.
Published in final edited form as: Nat Protoc. 2019 Jan;14(1):1–27. doi: 10.1038/s41596-018-0069-7

Fig. 2 |. Results from primary T-cell isolation and editing.

Fig. 2 |

a, Flow cytometry histograms depicting CD4 and CD25 levels on the cell surface of PBMCs (red), CD4+ T cells post enrichment (blue), and CD4+ T cells post stimulation with anti-CD3/anti-CD28/IL-2 (orange). After successful isolation and stimulation, the resultant cell population should be >95% CD4+ and >90% CD25+. Samples were run on an Attune NxT Flow cytometer and analyzed using FlowJo software v.10.1 (n > 100,000 events). b, Flow cytometry histogram depicting CXCR4 levels on the cell surface of primary T cells treated with non-targeting crRNPs (blue) or CXCR4-targeting crRNPs (red) relative to an unstained control (gray). After successful editing with CXCR4-targeting crRNPs, the resultant cell population should be <10% CXCR4+. Samples were run on an Attune NxT Flow cytometer and analyzed using FlowJo software v.10.1 (n > 100,000 events). c (Left), Chromatograms from Sanger sequencing of PCR amplicons over a target site in the CCNT1 locus in non-targeting treated cells (bottom) and in cells treated with CCNTI-targeting crRNPs (top). The gRNA target sequence and associated PAM are highlighted below. (Right) The TIDE output calculating the percentage of indels from these chromatograms. The total efficiency of indel generation provides a good estimate of knockout percentage in a cell population following crRNP treatment.