Supernatant of murine mast cells induces cytokine release and proliferation of EL4 cells. (A) EL4 cells and murine BMMCs were cultured for 1 week and the cytokines TNF, IL-6, IL-10, and IL-17A were measured in the supernatant of 2 × 10⁶ cells/mL using a cytometric bead array (CBA). Mean data of 4 separate experiments (n = 4) are presented as scatter grams with medians. (B) EL4 cells were cultured with or without supernatant of BMMCs for 48 hours, washed and incubated in medium for 48 hours. Levels of TNF, IL-6, and IL-17A were measured in the supernatant of 5 × 10⁵ cells/mL using CBA. Statistical significance was assessed by 2-tailed Student t test. (C) EL4 cells were cultured with (BMMC supernatant) or without (Control) supernatant of BMMCs or with a cocktail of several cytokines (IL cocktail; IL-1α, IL-1β, IL-2, IL-4, and IL-7) and proliferation was measured for 80 hours using Cell Titer 96 AQ_ueous One Solution Assay. Data represent the mean ± SD of 4 separate experiments (n = 4). When error bars are not shown, they were too small to be diagrammed. Statistical significance was assessed by 2-tailed Student t test (ns indicates not significant).