eBook: Cell and Gene Therapy - 11

a bright field and fluorescence live cell imaging assay.
A luminescence assay was used to evaluate the
functionality of the luciferase reporter cell lines in
which the cells were co-cultured with antigen-specific
CAR-T cells (ProMab) or mock CAR-T (ProMab)
cells derived from the same donor as a control at
Figure 2. CD19 CAR-T in vitro killing assay of Raji-Luc2 and WIL2-S-Luc2 measured using luminescence and live cell imaging. (A) CD19- positive
Raji-Luc2 cells (5 x 103
) or (B) WIL2-S-Luc2 cells (5 x 103) were seeded into a 96-well plate and were used as target cells for either CD19 CAR-T or
Mock CAR-T (control) from the same donor, which were seeded at various ratios of CAR-T cells to target Raji-Luc2 or WIL2- S-Luc2 cells (1:1, 2:1,
5:1, and 10:1). After 24 hours of co-culture, Bright-Glo was added to the indicated wells. The luminescence of the plate was read within 10 minutes
using a luminescence plate reader and determined to have a dose-dependent-specific killing with CD19 CAR-T cells that was greater than
the non-specific killing observed with mock CAR-T cells (* = significant difference and ns = not significant using unpaired t test, with a single
pooled variance). (C) Raji-Luc2 cells were stained with Vybrant DiO dye and real-time fluorescent imaging was measured every 30 minutes for
24 hours during the co-culture of Raji-LUC2 cells with CAR-T cells. (D) Two stained Raji-Luc2 cells (Green) from the co-culture experiment were
tracked for 6 hours and became surrounded by CAR-T cells, resulting in a decrease of fluorescence when treated with CD19 CAR-T as compared
to co-cultures with Mock-CAR-T cells. After 24 hours of co-culture, CD19 CAR-T cells showed a decrease in fluorescent cells as compared to 6
hours; in a co-culture with Mock CAR-T cells numerous Raji-LUC2 cells were present.
11
eBook: Cell and Gene Therapy

Table of Contents for the Digital Edition of eBook: Cell and Gene Therapy
