Cell cycle phase determination kit

The cell cycle phase was measured by DNA fragment staining using the cell cycle phase determination kit (Cayman Chemical, Ann Arbor, MI, USA) [2 (link)–6 (link)]. MDA-MB-231 (4 × 10⁵ cells/mL in a 24-well plate) were added to different concentrations of the OJEF for 12 h and then collected. After centrifugation, the precipitates were washed and resuspended in cell-based assay buffer. The cells were fixed and permeated by treating 1 mL of a fixative to each tube for around 2 h. After centrifugation, the fixatives were removed and the cell pellets were put into 500 μL of a staining solution (200 μL of RNase and 200 μL of PI), followed by leaving for 30 min at 20°C in the dark. Then, the cells were examined immediately by FACSCalibur flow cytometry.

A cell cycle analysis was performed by using the Cell Cycle Phase Determination Kit (Cayman chemical, Ann Arbor, MI, USA). The TOV112D (control), MAPT DNA‐transfected TOV112D, and MAPT siRNA‐transfected TOV112D cells were collected and centrifuged to pellet them at the bottom. After centrifugation, the cells were washed twice with an assay buffer and the cell pellet was resuspended to a density of 10⁶ cells/mL in the assay buffer. In order to fix and permeabilize the cells, 1 mL of fixative was added to each sample. Next, the fixed cells were centrifuged at 500 g for 5 minutes and the fixative was decanted. The cell pellet was suspended in 0.5 mL of staining solution and incubated for 30 minutes at room temperature. The cell cycle was analyzed in the FL2 channel of a flow cytometer with a 450 nm excitation laser (Becton, Dicknson and Company, Franklin Lakes, NJ, USA).

Cell cycle was determined using flow cytometry with a Cell cycle phase determination kit (Cayman Chemical, Ann Arbor, MI). For the cell cycle assay, 1 × 10⁵ cells were seeded in 6-well plate. Evaluation was done 24 hours after transfection of RECQL1-siRNA or control siRNA. Cells were harvested and fixed gently by adding 75% ethanol and placing at −20°C for 4–16 h. The cells were washed twice with PBS, resuspended in 300 μl PBS containing 100 μg/ml PI and 0.1 mg/ml RNase, incubated for 30 min at room temperature in dark, and analyzed using a flow cytometer. FlowJo 7.1.0 software (Tree Star, Ashland, OR) was used for data analysis and at least 10,000 cells were counted for each measurement. Experiments were repeated in triplicate.

Transfected 293T cells were stained with propidium iodide using the Cell Cycle Phase Determination Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. The stained cells were analyzed with a CytoFLEX (Beckman Coulter, CA) and Kaluza analysis software (Beckman Coulter).

Cell cycle phases were assessed by staining DNA fragments with PI using a Cell Cycle Phase Determination Kit (Cayman Chemical) according to the manufacturer's protocol. Cell cycle analysis was conducted similarly as performed previously (Ryu et al., 2018).