Synergy hybrid plate reader

CL-188 cells were seeded in a 12-well plate (6.4 × 10⁴ cells/well) in phenol-red-free medium essential medium with 10% FBS and 1% penicillin/streptomycin, and DLD-1 cells were in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. After overnight incubation, cells were treated with various concentrations of CUR and LUT or a combination of both (for instance, DMSO, LUT 30 μM, CUR 10 μM, CUR 10 μM + LUT 30 μM, CUR 15 μM, CUR 15 μM, and LUT 30 μM). The plate was incubated at 37 °C for 72 h. Images were photographed with the microscope; the cell proliferation assay was performed using the MTT vybrant assay kit according to the manufacturer’s instructions. In brief, the medium was aspirated from each well and replaced with 200 μL of fresh medium. Then, 50 μL of PBS-MTT was added to each well, after which the plates were incubated at 37 °C for 2 h. The SDS-HCl solution was added to each well, followed by incubation at 37 °C for 4 h. Absorbance was read at 570 nm using the synergy hybrid plate reader (BioTek Instruments Inc., Winooski, VT, USA. Part # 8041000). All experiments were separately repeated 5 times.

All strains were grown overnight in 3 ml of YPD at 30°C, with shaking at 250 rpm. Overnight cultures were used to seed fresh YPD cultures to an OD₆₀₀ of 0.15 and were grown at 30°C for 5 h. These mid-logarithmic cultures were then diluted to an OD₆₀₀ of 0.1 in a 100-μl final volume in a 96-well plate. Growth was assessed by measuring the OD₆₀₀ every 10 min for 20 h using a Biotek Synergy hybrid plate reader.

Total ATP was quantified with the bioluminescence-based Enliten ATP assay system (Promega, USA) as per the manufacturer’s instructions. Briefly, 100 μl aliquots of bacterial culture were collected at designated times and immediately heat inactivated and cell debris pelleted by centrifugation as described^{79 (link)}. Twenty-five microliter aliquots of cleared cell lysate were transferred into 96-well assay plates and analyzed according to the manufacturer’s instructions. Luminescence was detected using the luminescence mode in a Synergy Hybrid plate reader (Biotek Instruments, USA) and was expressed as relative luminescence units (RLU). Readings were normalized against CFU counts.