Cell cycle assay solution deep red

Cells were washed in PBS, resuspended in PBS, and stained with cell cycle assay solution (deep red; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) at 37 °C for 15 min. The cell cycle profiles were obtained using a FACSAria™ Cell Sorter at 640 nm. Data were analyzed using FlowJo software (Becton Dickinson).

HL-60 cells were plated at 8 × 10⁴ cells on a 35 mm dish (Sumitomo Bakelite, Tokyo, Japan) and chemicals were added. To observe the cell cycle, the cells were incubated for 1 d, and washed with PBS (500 µL) followed by incubation with Cell Cycle Assay Solution Deep Red (Dojindo, Kumamoto, Japan) at 37 °C for 15 min. Fluorescence signal was detected in FL4 using flow cytometer BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). For apoptosis/necrosis assay, the cells were incubated for 0.5–1 d, and washed with PBS followed by incubation with annexin binding buffer containing Annexin-V-FITC (MBL Life Science, Tokyo, Japan) and 7-Aminoactinomycin D (7-AAD) (BD Biosciences) at room temperature for 15 min. Fluorescence signals of FITC and 7-AAD were detected in FL1 and FL3, respectively, using flow cytometer BD FACSCalibur.

Cells were cultured in 6-cm dishes (200,000 cells/dish) to approximately 70% confluence and then treated with PG or Gly for 24 h. After treatment, cells were harvested using 0.025% trypsin and centrifuged for trypsin removal. Subsequently, cells were treated with Cell Cycle Assay Solution Deep Red (Dojindo Laboratories) and incubated for 15 min at 37 °C according to the manufacturer’s instructions. Data were obtained using FACSCelesta (Becton Dickinson, Franklin Lakes, NJ, USA) counting up to 10,000 cells and analysed using FlowJo software (Becton Dickinson).

Apoptosis was evaluated using the Annexin V-FITC Apoptosis Detection Kit (Nacalai, 15342-54), which includes an Annexin V-FITC conjugate and propidium iodide (PI) according to the protocol provided by the manufacturer, and was further analyzed using flow cytometry. For the cell cycle analysis, Cell Cycle Assay Solution Deep Red (Dojindo, C548) was used; the solution provided with the kit was added to the cell suspension, followed by incubation according to the manufacturer’s protocol, and a flow cytometric analysis. For staining, CD105-APC (eBioscience, 17-1057-42), CD90-PE (eBioscience, 555596), CD73-PE (eBioscience, 550257), CD44-PE (eBioscience, 550989), CD34-PE (eBioscience, 560941), CD31-Alexa Fluor^® 647 (BioLegend, 303112), and TRA-1-60-PE (Thermo Fisher, 12-8863-82) (1:50) was added to the cell suspension and incubated for 30 min before the flow cytometric analysis. BD FACSAria™ III was used to detect fluorescence. Results were plotted using FlowJo_v10.8.1 software.

Cells were trypsinized and fixed in cold (−30°C) 70% ethanol for two weeks. The fixed cells were washed in PBS and stained using the Cell Cycle Assay Solution Deep Red (Dojindo, Kumamoto, Japan) fluorescent probe. Cells were analyzed by flow cytometry using a FACS Canto II (BD Biosciences).

ARPE-19 cells incubated for 24 h after supernatant transfer in 24-well plates were treated with 1% Cell Cycle Assay Solution Deep Red (Dojindo, Tokyo, Japan) for 15 min at 37 °C to stain DNA. Flow cytometry analysis was performed on a FACS Canto II instrument and analyzed by using FlowJo software (BD, Franklin Lakes, NJ, USA).