Cycletest plus dna kit

Approximately 1.0 × 10⁶ cells in 6-well plates were treated with various concentrations of the indicated test samples at 37°C under 5% CO₂ for 48 h. Cell cycle analysis was performed by flow cytometry using the CycleTEST Plus DNA Kit (BD Biosciences), according to manufacture's instructions.

Cell cycle and apoptosis were analyzed by flow cytometry, and the transfected cells were harvested by trypsin digestion. An FITC-Annexin V Apoptosis Detection Kit was purchased from BD Biosciences. FITC-Annexin V and propidium iodide were used for double staining in accordance with the manufacturer’s instructions, followed by flow cytometry (FACScan; BD Biosicences, Franklin Lakes, NJ, USA). We first distinguished living cells, dead cells, early apoptotic cells, and apoptotic cells among the AsPC-1 and BxPC-3 cells. The relative proportion of early apoptotic cells in the transfection group and the control group was the target of our comparison. When analyzing the cell cycle, we calculated and compared the percentages of G0–G1, S, and G2–M phase cells in the transfected and control groups by FACScan analysis using a CycleTEST PLUS DNA kit (BD Biosciences) according to the instructions provided. All samples were assayed in triplicate.

WJ-MSCs were initially seeded at a density of 10,000 cells per 12-well plate (Falcon®, Corning, USA). Starting from Day 1 and continuing until Day 7, cells were harvested from three wells every day and counted using an automatic cell counter (Vi-Cell Blu, Beckman Coulter, Inc., USA). The cell counts obtained during the logarithmic growth phase were utilized to calculate the PDT using the listed formula. Further, on Day 3, WJ-MSCs were harvested specifically for the cell cycle assay. The Cycletest™ Plus DNA Kit (BD Biosciences, USA) was employed to determine the cell cycle, and the assay was conducted according to the manufacturer’s instructions. The proliferative index (PI) was calculated by the formula PI= (S + G2/M)/(G0/G1 + S + G2/M) [35 (link)].

Chloroquine (CQ), N-acetyl-L-cysteine (NAC) and acridine orange hemi (Zinc chloride) salt were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Caspase-8,-9, or -3 colorimetric assay kit and Z-VAD-FMK (a pan-caspase inhibitor) were obtained from R&D systems (MA, USA). Cycletest Plus DNA kit and FITC-Annexin V were purchased from BD bioscience Pharmingen (San Jose, CA, USA). Wortmannin and U0126 were purchased TOCRIS (Bristol, UK). 2’7’-dichlorofluorescein diacetate, BAPTA-AM, Ru360 and JC-1 were obtained from Calbiochem (San Diego, CA, USA). Dihydroethidium, lipofectamine 2000, anti-Alexa Fluor 488, Fluo4-AM and BAPTA were purchased from Invitrogen (Carlsbad, CA, USA). Rhod2 and Ruthenium Red were obtained from Abcam (Cambridge, UK). The antibodies used in this study are as follows; anti-caspase-3, anti-caspase-9, anti-caspase-8, anti-PARP, anti-ATG5, anti-total ERK, anti-p-ERK (Cell signaling Technology, MA, USA), LC3B/MAP1LC3B (NOVUS Biologicals, USA), anti-p62 lck ligand (BD bioscience Pharmingen, CA, USA), HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnologies, CA, USA).

Cells with AS-tDR-007872 or control mimics transfection were cultivated in the wells of 6-well plates for 24 hours and harvested by trypsinization. Cells were harvested post FITC-annexin V propidium iodide (PI) dual staining, followed by analysis via flow cytometry (FCM) (FACScan) and CellQuest both supplied by BD Biosciences, USA. For FCM, a Cycle TEST™ PLUS DNA Kit (BD Biosciences) was used. PI staining of cells was then performed by referring to the protocol and analyzed by FACS tank, for the calculation and comparison of the percentage of S-, G0/G1-, and G2/M-phase cells.

After 48 h of co-culture, 1.0 × 10⁶ cells were harvested and stained with Cycletest™ Plus DNA kit (BD Biosciences, USA) containing reagent such as trypsin, trypsin inhibitor, RNAse and propidium iodide for 10 min each. Cell cycle distribution was run using BD FACS CantoTM II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and further analyzed using BD CellQuest software (BD Biosciences, USA).