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. Author manuscript; available in PMC: 2016 Oct 31.
Published in final edited form as: Cell Rep. 2016 Oct 25;17(5):1453–1461. doi: 10.1016/j.celrep.2016.09.092

Figure 3. Efficient CD45 knock-out in human hematopoietic cells.

Figure 3

(A) Flow cytometry analysis of hCD45 expression in three AML cell lines and activated primary T cells 96 hours following electroporation with Cas9 only (blue) or Cas9/hCD45-sg1 RNP (red). (B) Effects of pre-culture before electroporation on gene disruption efficiency. hCD45 loss were examined in CD34+ cells cultured for 0, 24 and 48 hours in presence of cytokines before electroporation with Cas9/hCD45-sg1 RNP. hCD45 expression was evaluated 4 days after electroporation. Each experiment (n=8) was performed on CD34+ cells isolated from single donors. (C) Flow cytometry analysis of CD45 and CD34 expression in CD34+ cells 96 hours following electroporation with Cas9 only (left panel) or Cas9/hCD45-sg1 RNP (right panel). (D–E) Cell viability examined by trypan blue staining (D, n=8) or flow cytometry (E, n=6) of CD34+ cells electroporated either with Cas9 only (black) or with Cas9/hCD45-sg1 RNP (grey) relative to non-electroporated cells at the indicated time points. The cell counts (D) or viability (E) of Cas9 only and Cas9/hCD45-sg1 transfected cells were compared to the viable cell counts of non-electroporated cells. *, p<0.05; **, p<0.01; and ***, p<0.001 by non parametric t-test.