Flow Cytometry Sample Preparation

Sample pretreatment for flow cytometry analysis was conducted according to a previous study (Fang et al. 2006 (link)). Five milliliters of cells harvested from ePBR culture were pelleted and resuspended in 5 ml of ethanol/acetic acid (3:1) solution for fixation for 2 h at room temperature. After fixation, cells were rinsed twice and resuspended with 1 ml of FACS buffer containing 0.2M Tris–HCl (pH 7.5) and 20 mM EDTA and stored at 4°C. Prior to flow cytometry analysis, the cell density of each sample was measured with a Z2 COULTER COUNTER Analyzer (Cat. 6605700, Beckman Coulter), and all samples were normalized to 1 × 10⁶ cells/ml in a total 900 µl of FACS buffer, mixed with 100 µl of FACS buffer containing 1 mg/ml of RNase A, and incubated at 37°C for 2 h. For DNA staining, the RNase A-treated samples were rinsed once and resuspended in 1 ml of PBS buffer. Four hundred and ninety-five microliters of sample were mixed with 5 µl of 100-fold diluted SYTOX Green (cat. S7020, Thermo Fisher Scientific) for 5 min. Flow cytometry was performed with the LSR II Flow Cytometer System (BD Biosciences) at the MSU Flow Cytometry Core (https://facs.iq.msu.edu/; accessed 2022 February 7), and data were analyzed with FACSDiva Software (BD Biosciences).