Materials and Methods
Human squamous epidermoid carcinoma cells (A431) were grown to confluence in high glucose DMEM supplemented with L-glutamine, sodium pyruvate, penicillin/streptomycin, HEPES and 5% heat-inactivated FCS at 37°C and 5% CO2 in a humidified atmosphere (Binder CB incubator).
Incubation time study
Cells were harvested using trypsin/EDTA, then resuspended in fresh growth medium containing 5% FCS and seeded as a dilution series—ranging from 3,000 cells/well down to 35 cells/well—into a black 384 well tissue culture plate (100 µL/well). After overnight incubation at 37°C and 5% CO2 in a humidified atmosphere, 10 µl of the PrestoBlue reagent was added directly to the sample wells. The assay was incubated at 37°C and 5% CO2 inside the reader using the Infinite M200 PRO’s temperature control option and patent pending Gas Control Module (GCM™), which allows simultaneous control of O2 and CO2.3
Various incubation periods were tested to identify the minimum and optimum incubation times. After each incubation period (15, 30, 45, 60, and 75 mins), cell viability was determined by measuring the fluorescent signal in FI top mode. The whole experiment (incubation and detection) was carried out inside the microplate reader, without the need for any manual interaction. To provide maximum sensitivity, the system’s z-positioning with integrated background correction function was used to help overcome background fluorescence resulting from phenol red in the culture medium.4
The average value for each dilution was calculated (based on eight separate wells) and corrected by subtracting the average blank. The respective error bars were calculated using the Gaussian law of error propagation, and the theoretical limit of detection (LOD) was calculated using the following equation: