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. 2021 Aug 2;11:15648. doi: 10.1038/s41598-021-95157-6

Figure 3.

Figure 3

Spleen cells from animals treated with BCG are strongly activated in co-culture with infected MB49 cells. Spleen cells were obtained from mice submitted to the subcutaneous tumor model and treated with BCG (T) or control PBS (not treated—NT) (as shown in Fig. 1A) and euthanized at day 22 post-tumor injection. Splenocytes suspension was used for co-culture in vitro with infected MB49 cells (1 MB49 cell: 2 spleen cells). MB49 cells used for co-culture were previously infected with BCG (MB49 + BCG) for 24 h and washed to remove free BCG in the supernatant. Co-cultured cells were maintained together for 24 h (A–D) or 48 h (E), when the samples were collected for analysis. MB49 or splenocytes were also infected separately (not in co-culture) and used as controls. Measurement of TNF-α (A), IL-6 (B), IFN-γ (C) and CXCL10 (D) production in the supernatants after 24 h was performed by ELISA. (E) NO production was evaluated by the Griess method in supernatants after 48 h of stimulation. The values are representative of at least three independent experiments. *Statistically significant compared to MB49 + BCG, **P ≤ 0.01, ****P ≤ 0.0001. #Statistically significant data comparing treated (T) and not treated (NT), ####P < 0.0001.