Phenotypic and functional characterization of ethanol-treated tumor cells.

<p>(A) PANC/TGF-β were treated with 20% ethanol for 5, 10, or 15 min on ice and cultured for 48 h at 37°C in a 5% CO₂ humidified atmosphere. Ethanol-treated PANC/TGF-β were stained with FITC-conjugated mAbs against Annexin V and propidium iodide (PI). The entire scatter population (excluding obvious debris) was gated for most accurate analysis of cell changes. Cells undergoing early apoptosis are Annexin V positive and PI negative, and cells in late apoptosis or already dead (apoptotic death and necrotic death) are Annexin V and PI positive. (B) Ethanol-treated (20%, 15 min, on ice) or untreated tumor cells (PANC/Mock and PANC/TGF-β) were analyzed by flow cytometry for expression of HLA-ABC, HLA-A2 and MUC1 (solid area). The entire scatter population (excluding obvious debris) was gated. Unfilled histogram profile indicates the isotype control, and solid histogram indicates the specific antibody. (C) Mean concentration of the active form of TGF-β1, IL-10, VEGF, and HSP90α by ethanol-treated (▪) and untreated (□) tumor cells (PANC/Mock and PANC/TGF-β) (1×10⁴/mL) was analyzed by ELISA. (D) PANC/TGF-β were treated with 20% ethanol for 5, 10, or 15 min on ice and cultured for 48 h at 37°C in a 5% CO₂ humidified atmosphere. The entire scatter population (excluding obvious debris) was gated. Ethanol-treated and untreated PANC/TGF-β were analyzed by flow cytometry for expression of calreticulin (left panel), CD31 (middle panel), and CD47 (right panel). Untreated PANC/TGF-β were used as controls. Solid histogram indicates the specific antibody. (E) Mean concentration of HMGB1 by PANC/TGF-β treated with 20% ethanol for 5, 10, or 15 min on ice or untreated PANC/TGF-β (1×10⁵/mL) was analyzed by ELISA. The mean ± SD of three experiments is shown. ***<i>P</i><0.001; **<i>P</i><0.01; *<i>P</i><0.05.