Cck 8 kit

Cell proliferative ability was detected using a CCK-8 kit (Merck) according to the protocol. After treatment, 1-2 × 10³ cells were cultivated for four days, and 10 μl of the CCK-8 kit was added. Thereafter, the samples were incubated for two hours in the dark at 37 °C with 5% CO2. 450 nm OD value was detected with a microplate reader. As for the Colony formation assay, cells (500 or 1000 cells) were cultured with a complete medium for 7 to 14 days. Cells were then washed with PBS, and fixed for 30 min and stained with 0.1% crystal violet solution.

Cells were seeded in duplicates on two identical 96-well plates at 5000 cells per well. To test the oxidative stress management, cells were treated with either 0 or 100 μM H₂O₂. The first plate was then treated with 10% WST-8 with the CCK-8 kit (Merck), according to manufacturer’s instructions and incubated for 2 h at 37°C. The absorbance was then measured at 460 nm. We proceeded the same way with the second plate 4 days later. Cell viability was determined subtracting absorbance values of day one from the respective absorbance values of day four.

TU212 cells (5 × 10³/well) were inoculated and cocultured with macrophages treated with exosomes from HOK and TU212 cells or transfected macrophages with a 24, 48, or 72 h CCK-8 kit (Sigma, USA). A spectrophotometer (Molecular Devices, San Jose, USA) was utilized to measure the absorbance (OD) at 450 nm.

Briefly, cells were cultured in 96-well plates and stimulated with different concentrations of ROT, AA, Mdivi-1or Dynasore for 24, 48, or 72 h. The cell viability of each treated sample was measured using the CCK-8 kit (CCK-8, Sigma 96992, St. Louis, MO, USA) according to the manufacturer’s instructions. The absorbency of cells was measured using a 96-well plate reader at 450 nm.

Cell proliferation assay was performed with and without E₂ treatment for both Vec and JOE cells. In brief, cells were starved in E₂ depleted media for 3 days and plated at a density of 10⁴ cells per well (Day 0). 10 nM E₂ was added after 24 h and media was replaced each day. Cells were counted in Hemocytometer at time points mentioned in the figure. Cell viability assay was performed by CCK8 kit (Sigma Aldrich, USA). Briefly, cells were starved for 3 days in E₂ deprived media at a density of 10⁴ cells/well. Different doses of 4-OHT as indicated were added and cells were incubated for 72 h. The media was changed every day and absorbance was measured at 450 nm.

The seeding of cells in their log growth phase (3 × 10³ cells/well) was done and maintained in tissue culture plates (96‐well). Assay for cell proliferation was done using the CCK‐8 kit from Sigma at specific time‐points as per instructions. Assay for colony formation was carried out as per a previously published protocol.²⁹, ³⁰ In brief, cells were plated independently in the wells of tissue culture plates (6‐well). After 2 weeks, colonies that were visible were paraformaldehyde‐fixed (4%) for 20 minutes and stained (crystal violet; 0.1%) for 60 minutes. The efficiency of colony formation was determined as the total colonies with diameter > 0.5 mm. Proliferation assay using EdU (5‐ethynyl‐2'‐deoxyuridine) was done using the kit Molecular Probes EdU‐Alexa imaging from Life Technologies. After two days of transfection, cells were incubated for 60 minutes with EdU (10 μmol/L), followed by fixing, permeabilization and staining with reaction cocktail AlexaFluor 594 and Hoechst 33342 for EdU and cell nuclei, as per provided protocol, and then visualized and the image was acquired under a fluorescent microscope. Each assay was carried out at least thrice.

Curcumin, CCK-8 kit, DMSO, and N-acetyl cysteine were purchased from Sigma Chemical Co (St. Louis, MO, United States) (Caspase-9, caspase-3,cleaved caspase-3,PARP,XIAP,p-Akt,Akt,GSK3,P-GSK3,FOXO1,P-FOXO1,GAPDH,cIap1,cIap2, Bcl-2, Bcl-xl, caspase 8, and cleaved caspase-8 antibodies were obtained from Cell Signaling Technologies (Beverly, MA, United States). Bax, p-H2AX, and cytochrome c antibodies and cisplatin were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V fluorescein isothiocyanate (FITC), Propidium Iodide (PI), and p-H2AX (pS139) antibodies were purchased from BD Biosciences (San Jose, CA). z-VAD-FMK was obtained from Calbiochem (San Diego, CA, United States). CellROX Green was obtained from Invitrogen (MA, United States). Curcumin was dissolved in DMSO and further diluted in the cell culture media for the treatment of cells, so that the final concentration of DMSO in wells is 0.1% at the highest concentration of Curcumin used in the study. Viability assays showed that 0.1% DMSO is non-toxic to the cells (data not shown).