Flow cytometry

A Cell Cycle Detection Kit was used to detect the cell cycle distribution. Appropriate number of cells were seeded into 6-well plates and then treated with POS at the prescribed concentrations for 24 h. The cells were fixed with 70% (v/v) cold ethyl alcohol at 4°C for overnight and stained with 500 μL RNase A/PI staining solution (v/v, 1:9) in the dark at room temperature for 30 min. The cell DNA content was tested by flow cytometry (Sysmex, North Rhine-Westphalia, Germany) and analyzed with FCS Express software version 6.

Cells were seeded in 6-well plates overnight and treated with DMSO or POS as indicated. Then, the treated cells were digested, harvested, and stained with acridine orange (AO) solution (1 μg/ml). After washing with PBS, the cells were detected by flow cytometry (Sysmex). The results were handled by FCS Express software version 6.

Untreated TH1 cells, P-cresol-treated TH1 cells, and P-cresol-pretreated TH1 cells after treatment with si-CK2α or si-Scr were trypsinized and fixed with 70 % ethanol at -20 °C for 2 h. Next, the groups were incubated with RNase and the DNA-intercalating dye propidium iodide (PI; Sysmex) at 4 °C for 1 h. PI-stained groups were characterized by flow cytometry (Sysmex). Events were recorded for at least 10,000 cells per sample. The sample data were analyzed using the FCS express 5 software (DeNovo Software). Independent experiments were carried out thrice.

The Kio sperm-like cells were blown down gently by PBS. After being filtered through a 40 μm cell strainer, the cells were incubated with DAPI (Invitrogen) for about 15 min, then examined by flow cytometry (Sysmex-partec, Germany). As previously described, specific methods were carried out (Peng et al., 2020 (link)).

Cells were incubated with Fluo-3 AM (S1056, Beyotime) or Rhod-2 (Abcam, ab142780) for 30 min. After washing with PBS, the intensities of cytoplasmic Ca²⁺ and mito-Ca²⁺ were detected by flow cytometry (Sysmex Partec GmbH, Germany) and immunofluorescence using a Leica confocal microscope (Leica, Wetzlar, Germany). Cytoplasmic ROS was measured by DCFH-DA (Sigma) (Domingues et al., 2020 (link)). ImageJ was used to assess fluorescence intensity (Fluo-3, Rhod2, and TMRM). Data (F/F0) were obtained by dividing the fluorescence intensity (F) by (F0) at the resting level (t = 0), which was normalized by the control group. The TUNEL assay was used according to the manufacturer’s instructions to detect cell death after hypoxic injury. For quantification, the numbers of TUNEL-positive cells were calculated in at least 10 random separate fields to obtain the average percentage in different groups (di Somma et al., 2020 (link)).