Bricyte e6 flow cytometer

Cellular reactive oxygen species (ROS) level was measured by a technique that converts carboxy-H₂DCFDA (5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, Sigma Aldrich) into a green fluorescence dye 2′,7′–dichlorofluorescein (DCF). Briefly, cultured cells were counted, washed with phosphate-buffered saline (PBS), and resuspended in carboxy-H2DCFDA at a final concentration of 10 μM in regular culture medium with reduced serum (2%). Cells were incubated in the dark for 30 min in a conventional incubator at 37 °C. After the incubation period, cells were washed with PBS, seeded in six-well plates at the density of 3 × 10⁵ cells/well, and treated with ME at a final concentration of 10 µg/mL in regular culture medium with reduced serum (2%). These cells were cultured for 24 h at different thermal conditions. Next, the cells were collected as described above, washed twice with PBS, and resuspended in PBS. ROS level was assessed by immediately analyzing cells using flow cytometry (FL1 channel/green fluorescence) BriCyte E6 flow cytometer (Mindray, Shenzhen, China). Results are presented as a percent change in comparison to control cells (cultured at 37 °C and not treated with ME).

The cell cycle was determined by the quantitation of DNA content using the nucleic acid stain propidium iodide followed by flow cytometry analysis. Propidium iodide (PI) is a fluorescence dye that binds both types of nucleic acids, DNA and RNA, proportionally to the amount of material present in the nucleus. Cells were seeded at a density of 3 × 105 in six-well plates, as described above. Following overnight preincubation, cells were treated with ME and exposed to various temperature conditions for 24 h. After treatment, the culture media was removed, and cells were collected as described above. Cell cycle analysis was carried out using a CellCycleFlowEx^® Kit (Exbio, Vestec, Czech Republic) according to the manufacturer’s instructions. Cells were stained for 30 min with PI, and RNA was digested using RNAse. Next, flow cytometry analysis was conducted within 4 h using a BriCyte E6 flow cytometer (Mindray, Shenzhen, China). The cell cycle distribution was presented as the percentage of cells containing 2n (G1 phase), 4n (G2 and M phase), and between 2n and 4n (S phase), as determined by PI staining.

CCK-8 (ZETA LIFE, Menlo Park, CA, USA) was used to monitor cell proliferation per the manufacturer’s instructions. Approximately 2 × 10³ cells were seeded into a 96-well plate, 10 μL CCK-8 solution was added to each well, and the cells were incubated at 37 °C for 2 h. The OD was then measured using a Multiskan FC-type microplate reader at 450 nm (n = 3, three technical repetitions were performed for each sample). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (Multi Sciences, Hangzhou, China) was used to detect cell apoptosis per the manufacturer’s instructions. After washing with PBS, the collected cells were resuspended with 1 × binding buffer. Next, 5 μL Annexin V-FITC and 10 μL PI were added, mixed, and incubated at room temperature for 5–10 min in the dark; 400 μL 1 × Binding Buffer was then added and mixed, and the samples were filtered with a 300-mesh cell sieve and tested on a BriCyte E6 flow cytometer (Mindray, Shengzhen, China) (n = 3, three technical repetitions were performed for each sample).

Yeast cultures were incubated for 96 h and 300 μL samples were collected at 24 h intervals. Prior to flow cytometry analysis, 20 uL of each cell culture was diluted in 980 uL of Phosphate-Buffered Saline (PBS), pH 7.4. Flow cytometry analysis was performed in BriCyte E6 flow cytometer (Mindray Bio-Medical Electronics, Nanshan, China), and the data were analyzed using FlowJo™ v10 (BD Life sciences, Franklin Lakes, NJ, USA). Appropriate gates were applied in order to select single cells (Supplementary Figure S1). The geometric mean of the generated data was used to calculate the fluorescence intensity of each sample. The number of events recorded was set to 50.000. For the fluorescence microscopy, cells were centrifuged at 5000× g for 2 min, washed with dH₂O, and resuspended in PBS before being imaged with the ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, CA, USA).