Wallac plate reader

MTT cell viability assay was performed to evaluate the cytotoxicity of the curcumin-loaded PCL scaffolds [29 (link),31 (link),32 (link)]. Briefly, cells were plated on a 96-well plate at a density of 5 × 10³ cells per well in 200 µL culture media and incubated for 24 h under 5% CO₂ at 37 °C. After 24 h, the tested samples (100 µL) were added into individual wells and incubated for 48 h, along with negative controls including untreated cells (control 1) and cells treated with blank release media (control 2). After 48 h, cells were rinsed gently with PBS, and fresh media was added along with 10 μL of MTT solution (5 mg/mL in sterile PBS, pH 7.40). All plates were incubated for a further 4 h. The MTT solution was then removed from each well and 100 μL of DMSO was added to dissolve any MTT formazan crystals. Plates were covered and left on a plate shaker for 5 min. The absorbance of formazan product was then taken at 570 nm on a Perkin Elmer Wallac plate reader (Perkin Elmer, Inc., Waltham, MA, USA). The results were expressed as percentage viability and groups treated with curcumin were compared to the untreated group (control 1).

An 8-point serial dilution (50000 µM, 12500 µM, 3125 µM, 781 µM, 260 µM, 65 µM, 16 µM, 4 µM) in DMSO was prepared for each test compound. HeLa cells were grown at 37 °C in 5% CO₂ and 50:50 DMEM:F12 medium (GIBCO) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). In each well of a 384-well plate, 20 µl of fresh medium was added, .5 µl of drug stock was then plated in each well for a final drug concentration of 50 µM to 0.04 µM, and 30 µl of 5 × 10⁴ cells/ml cell suspension was dispensed into each compound containing well in triplicate. Plates were incubated at room temperature for 30 minutes and placed at 37 °C for 72 hours. 25 μl of CellTiterGlo® reagent (Promega) was dispensed into each well. Plates were incubated at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was measured using a Wallac plate reader (PerkinElmer).

20 µl of fresh DMEM:F12 medium was added to each well of a 384-well plate, 0.5 µl of drug stock was then plated into each well for a final drug concentration of 50 µM, and 30 µl of 5 × 10⁴ cells/ml HeLa cell suspension was added. Plates were incubated at room temperature for 30 minutes and then placed at 37 °C for 72 hours. After equilibrating at room temperature for approximately 30 minutes, 25 μl of CellTiterGlo® Reagent (Promega) was added to each well. Plates were incubated at room temperature for 10 minutes to stabilize the luminescent signal and luminescence was recorded using a Wallac plate reader (PerkinElmer). The average readout for the control DMSO-treated cells was used to calculate the average % cell viability of compound-treated cells. Similarly, HCT116, U2OS, and A549 cells were treated with the EC₉₀ of each of the six selected drugs and cell viability was measured as described above.