Fxcycle far red stain

For cell cycle analysis, Chaga mushroom-treated cells were labelled with EdU (Thermo Fisher Scientific) for 2 h before harvesting. Cells were collected and stained with the Click-iT Plus EdU Alexa Fluor 488 flow cytometry kit (Thermo Fisher Scientific) following the manufacturer’s instructions. After washing, DNA was stained with FxCycle™ Far Red stain (Thermo Fisher Scientific) according to the manufacturer’s protocol. The cells were then acquired on a BD Accury C6 Plus (BD Bioscience, Franklin Lakes, NJ, USA), followed by analysis using FlowJo software (BD Bioscience). For apoptosis analysis, the cells were seeded at a density of 5 × 10⁵ cells in a 60 mm culture dish. After 24 h, the cells were treated with various concentrations of Chaga mushroom extract (0, 160, 200, 400, and 800 µg/mL) for 6 h, harvested by trypsinization, and collected by centrifugation. The cells were washed with cold PBS, resuspended in 100 µl Annexin V binding buffer, and stained with 5 µl FITC-conjugated Annexin V and 5 µl propidium iodide in the dark for 20 min at room temperature. After incubation, 400 µl binding buffer was added and apoptotic cells were analyzed according to the manufacturer’s instructions (Annexin V-FITC apoptosis detection kit, BD Bioscience) using a BD Accury C6 plus flow cytometer (BD Bioscience). Cell distribution was analyzed using FlowJo software (BD Biosciences).

A total of 1 million cells were fixed in 1 mL of 2% (w/v) paraformaldehyde (Fluka) in Dulbecco’s phosphate-buffered saline (DPBS) on ice for 15 min, washed with DPBS twice by centrifugation at 800 g for 5 min, resuspended in 1 mL of 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in DPBS supplemented with 1% (v/v) FBS, treated with 0.1 mg/mL RNase A (Thermo Fisher Scientific), and stained with 1 μL of FxCycle Far Red Stain (Thermo Fisher Scientific) at room temperature for 30 min. Cell cycle profiles were modeled with the Cell Cycle function in FlowJo v10.0.8 [Watson (Pragmatic); CV of G2 peak = CV of G1 peak].

Cell cycle analysis was performed 72 h post- transfection or drug treatment. Approximately 1 × 10⁶ cells were pelleted, washed in phosphate-buffered saline, and fixed in 75% ethanol overnight at 4°C. Cells then were washed in phosphate-buffered saline, re-suspended in a 1× saponin-based permeabilization buffer and wash reagent (Thermo Fisher Scientific, Waltham, MA, USA), and stained with FxCycle™ Far Red stain (Thermo Fisher) for 30 minutes at room temperature. Samples were evaluated on a FACSCalibur cytometer (Becton Dickinson, Mountain View, CA, USA). Data were analyzed using FlowJo 7.6.5 software (Tree Star Inc., Ashland, OR, USA).

For flow cytometry (FCM), cells treated with 5-H-Y or cisplatin for 24 h were pulse-labeled for 60 min with 10 μM 5-ethynyl-2′-deoxyuridine (EdU). The dead cells were washed away prior to the cell harvest. After harvesting, to fluorescently label the incorporated EdU in newly synthesized DNA, we used Click-iT EdU Flow Cytometry Assay kits (Invitrogen). The cells were also stained with FxCycle Far Red Stain (Invitrogen) to stain DNA. FCM analysis was performed with a JSAN cell sorter (Bay Bioscience) using a logarithmic FL1-A channel for EdU detection and a linear FL5-A setting for FxCycle Far Red Stain. The cells with abnormal shapes or multiple nuclei were eliminated by forward/sideward scatter (FSC/SSC) gating. Analysis was performed using the Flowlogic software. For each analysis, we started with ~10⁶cells and ~10⁴cells of the flow cytometer result were plotted.