Fig. 3
(A) Diagram outlining the steps to prepare and co-culture DC-CIK cells with GBOs with downstream analyses. The medium and culture conditions are indicated below each step. CM, CIK cell medium; GM, GBO medium; RH, relative humidity. (B) Phase contrast images of individual GBOs at 0, 24, 48, and 72 h, co-cultured with DC-CIK cells with or without a transwell system, and a control group of GBOs alone. Representative images are based on the analysis of 10 GBOs. Scale bar, 500 μm. (C) Sample phase contrast images of the edge of GBOs when transwell co-cultured with DC-CIK cells for 72 h (E: T 10:1 W/T transwell E: T 1:1 W/O transwell). Representative images are based on the analysis of 10 GBOs. (D) Quantification of IFN-γ in media from co-cultures with DC-CIK cells, with or without a transwell system, was performed using ELISA at 0, 24, 48, and 72 h. Values represent mean ± s.e.m. (n = 3 GBOs per sample per timepoint). (E) Sample confocal images of fluorescent immunohistology for cleaved caspase 3 (CC3) and cluster of differentiation 3 (CD3) in GBOs after co-culture with either CIK cells for 48 h (E: T 1:5), showing increased apoptosis when co-cultured with CIK cells, with higher-magnification images of boxed regions highlighting apoptotic cells near CIK cells at the GBO periphery and core. Representative images are based on the analysis of five GBOs. Images were taken using Zen 2 Blue software and were processed using Illustrator CC, ImageJ, Imaris and/or Photoshop CC software.
