Cell titer 96 aqueous solution

To examine the antiproliferation effect of the study agents, HCT-116 cells were seeded in 96-well plates at approximately 1 × 10⁴ cells/well. After 24 h, indicated concentrations of drugs were added to the wells. After treatment for 48 h, cell proliferation was evaluated using an MTS assay according to the manufacturer’s instructions. Briefly, the medium was replaced with 100 μL of fresh medium and 20 μL of MTS reagent (CellTiter 96 Aqueous Solution, Promega, Madison, WI, USA) in each well, and the plate was returned to the incubator for 1–2 h. A 60 μL aliquot of medium from each well was transferred to an ELISA 96-well plate and its absorbance at 490 nm was recorded.

Cell viability was measured using the Cell Titer 96 Aqueous Solution (Promega). In brief, cells were culture in 96‐well plates and treated with RPE for 24 hr. The 100 mg/ml of RPE stock sample was treated to the cultured media as indicated concentration (50–1,000 μg/ml). Following the incubation period, 20 μl MTS solution was added, and cells were further incubated for 1 hr. Cell viability was measured with absorbance at 490 nm using a microplate reader (Infinite^®2000 PRO, Tecan, Switzerland).

The comparative antiproliferative assay was conducted using the MTS cell viability method to evaluate the potency of the conjugates to inhibit the proliferation of MCF-7 breast cancer cells and HEK-293 normal embryonic kidney cells. The cells were seeded (5000/0.1 mL) in each well using a 96-well plate. The MCF-7 cells were treated with PTX and CPT drugs and their corresponding conjugates in the medium. The final concentrations of all the compounds were adjusted to be 5 μM. Cells were incubated with the treatments for 4 h. Then, the treatments were removed and replaced by the fresh medium and incubated for a further 72 h. A similar treatment was followed for the HEK-293 cell line with PTX1, PTX, and cyclic peptide [W(WR)₄K(βAla)] at 5 μM. The CellTiter 96 aqueous solution (Promega, Madison, WI, USA) was used to measure the cell viability based on the fluorescence intensity of them at 490 nm. Here, SpectraMax M2 microplate spectrophotometer was employed for the assay. The cell viability was calculated relatively based on the cell survival as [(OD value of cells treated with the test mixture of compounds) − (OD value of culture medium)]/[(OD value of control cells) − (OD value of culture medium)] × 100%.

Cell viability was measured by the MTS method following the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, 100 µL of cell suspension was collected from the circulation system and then added to 1 well of a 96-well plate. After incubation for 12 h, 20 µL of sterile CellTiter 96 aqueous solution (5 mg/mL; Promega, Madison, WI, USA) was added to each well, and the plate was incubated for 4 h at 37 °C. The absorbance of the cell solution was measured at 490 nm using a Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA).

Cells were plated at 2.5 × 10⁴ cells/well and left for 24 h to adhere to the 96-well plate. Drug combinations were freshly prepared in media and were added for 24 h. During the last 4 h of incubation, Cell Titer 96 Aqueous solution (Promega) was added. The absorbance at 490 nm was measured using a Flexstation 3 plate reader.

Cell viability was assessed using the Promega Cell Titer96 aqueous solution (G3580, Madison, WI, USA), as previously described [18 (link)]. On the other hand, the cell death assays were performed through Annexin V-FITC (fluorescein isothiocyanate)/7AAD (BD Pharmingen) analysis. The number of dead cells was analyzed by counting the number of cells that stained positive for Annexin V-FITC and 7-ADD.

The cell proliferation assays were conducted in adherent cell lines (HCT116, MCF-7, MDA-MB-468, HS578T, OV-2008, A2780, SKOV3, 786-O, HEK-293, and HeLa) by using CellTiter 96 AQueous non-radioactive colorimetric method (G5421, Promega, Madison, WI, USA). Briefly, a total of 3000 cells/well were seeded in a 96-well flat-well plate followed by treatment with FD-895 (100 nM to 2.0 μM), pladienolide B (100 nM to 2.0 μM), cisplatin (1 μM to 30 μM) or etoposide (1 μM to 30 μM) in triplicate for 48 h at 37°C. Following the incubation, 20 μL of CellTiter 96 AQueous solution (Promega, Madison, WI, USA) was added directly to each well. Non-treated cells were considered as the control. After staining, the plates were incubated for an additional 2 h and then read on a 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance readings were recorded absorbance at 490 nm using empty wells (air) for background collection.