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. 2020 Jul 14;10:1143. doi: 10.3389/fonc.2020.01143

Figure 2.

Figure 2

Preparation of the third generation anti-HER2 CAR-T cells. (A) Diagram of anti-HER2 CAR. (B) After activation and separation, mouse spleen lymphocytes were detected by anti-CD3 monoclonal antibody using flow cytometry. The values indicated the percentage of CD3-positive mouse spleen T cells. The left histogram was an isotype control. (C) After the mouse spleen T cells were transduced with the recombinant lentiviruses at MOI 20 for 4 days, the percentages of 7-AAD-negative (viable) cells and GFP-positive (transduced) cells were analyzed using flow cytometry, with (untransduced) blank T cells as the control. The value indicated the percentage of transduced viable cells. (D) The expression level of CAR (containing exogenous CD3ζ) in CAR-T cells was detected by western blotting using an anti-CD3ζ antibody, with blank T cells as negative control, and endogenous CD3ζ as a loading control. The experiments have been repeated for three times. CAR, chimeric antigen receptor; scFv, single chain variable fragment; VH, heavy chain variable region; VL, light chain variable region; MOI, multiplicity of infection; 7-AAD, 7-aminoactinomycin D; Blank T, T cells not transduced; CAR-T, T cells transduced with CAR.