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5 cells was re-suspended in 95 μL PBS, then 5 μL 0.01% acridine orange (Sigma) was added. Cells were mixed at room temperature, and then a drop of cells was enveloped with a coverslip on the slide and observed under a fluorescence microscope.
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6 cells were re-suspended and fixed in 70% ice-cold ethanol overnight at 4 °C. After brief centrifugation, cells were washed with PBS and incubated for 30 min at 37 °C with 1 mL propidium iodide (PI, Sigma) solution (50 μg/mL PI, 50 μg/mL RNase, 10 μL/mL Triton X-100, 1 mg/mL Sodium citrate). Samples were analyzed using a FAC Scan flow cytometer (Becton Dickinson, US). ModFit software (Verity Software House, Inc.) was used to analyze the data for 20,000 cells in each test.
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