Annexin 5 flous

After exposure to hyperthermia, the cells were re-incubated at 37°C for 0 h, 24 h, or 48 h. Then, the cell cycle phases were analyzed. Briefly, the cells were fixed with 70% ethanol at 4°C overnight. After washing with Ca²⁺-Mg²⁺-free Dulbecco's PBS, the cells were treated with 0.1 mg/ml RNase (Type I-A; Sigma, St Louis, MO, USA) and then stained with 100 µg/ml propidium iodide (PI; Sigma), in the dark, at room temperature for 20 min. After passing through a 40 nm nylon mesh, the samples were kept on ice until measurements. The data obtained, using the FACS calibrator, were used to analyze the cell cycle phase proportions with ModFit software. A cell fraction of DNA, below the sub-G0/G1 peak, indicated apoptotic cells; DNA histograms were used for their estimation.
For Annexin V staining, the cells were directly stained with PI and Annexin-V Flous (Roche) for 10 min and then washed with incubation buffer. The cells were identified with a FACS calibrator after setting the voltage using non-treated stained control cells. The cells were analyzed using Cell Quest software and classified into four different stages: unstained living cells, early age apoptotic cells stained only with Annexin-V, middle age apoptotic cells doubly stained, and late age apoptotic and necrotic cells stained with PI only.

The cells were harvested, washed twice in PBS and resuspended in 100 μL annexin V incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl₂) containing 1% annexin V FLOUS (Roche Molecular Biochemicals) and 500 μg/μL PI stain. The samples were incubated for 15 min at room temperature followed by adding 400 μL of ice-cold annexin V incubation buffer and subsequently analyzed on a cytometry machine.