Molecules and Cells

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Fig. 3.

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Fig. 3. (A) Splenocytes, obtained from BALB/c pre-immunized with CT26, were mixed and cultured with MMC-inactivated tumor clones expressing IL-15 or IL-15:IL-15Rα. The ratio of splenocytes to tumor clones is 50:1. After 48 h, the morphology of MMC-inactivate tumor clones in the mixed-cell cultures was recorded under a microscope. A representative result was shown from 3 independent experiments. (B) Pre-immunized splenocytes were stimulated in vitro with MMC-inactivated wild-type cells (antigen sources) and culture supernatants of tumor clones expressing IL-15:IL-15Rα complexes (cytokine sources) for 48 h. The culture supernatant from a parent or mock-transfected clones was used as control. The stimulated splenocytes and CFSE-labeled wild-type CT26 target cells were mixed for at ratio of 20:1 or 50:1. After 6 h incubation, the cell mixtures were collected and stained with PI. The percentage of CFSE+PI+ cells (dead cells) were counted by FACS. (C and D) The activated splenocytes, as described in (B) were pre-incubated with 10 μg/ml anti-CD4 or anti-CD8 antibodies for 30 min before mixing with CSFE-labeled CT26 target cells. After 6 h incubation, the percentage of CFSE+PI+ cells were analyzed by FACS. (E) The activated splenocytes, as described in (B) were mixed with CFSE-labeled YAC-1 target cells (ratio 50:1). After 6 h incubation, the cells were harvested and stained with PI. CFSE+PI+ cells were counted by FACS. *P < 0.05; **P < 0.01; ***P < 0.001.
Mol. Cells 2019;42:869~883
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