Stressed apoptotic tumor cells express membrane HSPs.
(A) Nonstressed or heat-stressed (42°C, 1 hour) 12B1-D1 cells were treated with AP20187 for 4 to 6 hours and then washed. Surface expression of specific antigens was determined by incubating with anti-HSP72, anti-HSP60, or anti-HSP90 monoclonal antibodies followed by staining with Cy3-conjugated affiniPure F(ab′)2 fragment goat antimouse IgG. Stained cells were washed and further stained with annexin V–FITC. Cells were then washed and transferred onto microscopic slides using cytospin centrifugation followed by examination under confocal laser microscopy. Original magnification × 63, oil objective. (B) For flow cytometry, the secondary antibody was F(ab′)2 fragment goat antimouse IgG conjugated with ALEXA FLUOR 488. Following staining with secondary antibody, the cells were further stained with the vital dye propidium iodide (PI) and analyzed by flow cytometry (12B1-D1/AP indicates 12B1-D1 cells treated with AP20187; 12B1-D1/HS/AP indicates heat-stressed 12B1-D1 cells treated with AP20187).
