Supernatant of mast cells induces cytokine release and proliferation of PCL cells. (A) HMC1 cells (1 × 10⁶) were treated with medium (black circles), calcium ionophore A23187 (black triangles) or cromolyn (diamond) for 30 minutes at 37°C, washed and incubated in medium for 6 hours. Mast cell products IL-6, TGF-β1, VEGF, CCL2, and CXCL8 were measured in the supernatants using CBA. Mean data of 4 separate experiments (n = 4) are presented as scatter grams with medians. Statistical significance was assessed by 2-tailed Student t test. (B) Mac2B and SeAx cells were cultured with or without supernatant of HMC1 cells for 24 hours, washed and incubated in medium for 6 hours. IL-6 mRNA levels relative to GAPDH expression were determined using quantitative real-time PCR. Statistical significance was assessed by 2-tailed Student t test. (C) Mac2B and SeAx cells were cultured with or without supernatant of HMC1 cells for 48 hours, washed and incubated in medium for 48 hours. Levels of IL-6 were measured in the supernatant of 5 × 10⁵ cells/mL using CBA. Statistical significance was assessed by 2-tailed Student t test (ns indicates not significant). (D) Primary T-cells, Jurkat cells, primary Sézary cells, and the cell line SeAx were cultured with (HMC1 supernatant) or without (Control) supernatant of HMC1 cells or with a cocktail of several cytokines (IL cocktail; IL-1α, IL-1β, IL-2, IL-4, and IL-7). SeAx cells were additionally cultured with mast cell supernatant derived from HMC1 cells treated with cromolyn (Cromolyn). Proliferation was measured for 80 hours using the 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).