September 15, 2013 (Vol. 33, No. 16)
Christian Oberdanner, Ph.D. Tecan
Study Performed with Tecan’s Infinite® Multimode Microplate Readers
Assessment of cellular viability is vital for laboratories working with in vitro cell-based systems. Resazurin is a fluorescent redox indicator dye that is frequently used to determine viability of cells directly in culture, with only viable cells able to convert the dark blue oxidized form of the dye (resazurin) into a red-fluorescent reduced form (resorufin; λEx = 570 nm; λEm = 590 nm). Life Technologies offers an enhanced version of resazurin, the PrestoBlue Cell Viability Reagent, which allows significantly shorter incubation times (minimum 15 mins vs. 1 hr) for increased experimental efficiency and performance.1
This application note describes the evaluation of the PrestoBlue Cell Viability Reagent using the Infinite M200 PRO and the Infinite M1000 PRO multimode microplate readers. These instruments are based on Tecan’s Quad4 Monochromators™ and Premium Quad4 Monochromators technologies respectively, and offer a number of innovative features designed to support cell-based assays.2
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:
For the average sample value, the result for the first dilution of the series (3,000 cells/well) was used.
In a second experiment, cells were harvested using trypsin/EDTA, then resuspended in fresh growth medium containing 5% FCS and seeded into a black 96-well tissue culture plate with a density of 12,000 cells/well (200 µL/well). The supernatant was removed and 200 µL DMEM containing varying concentrations (0.015–250 nm) of staurosporine (STS) was pipetted into each well and incubated overnight at 37°C and 5% CO2.
Following incubation, cells were stained by adding 20 µL PrestoBlue reagent directly to the sample wells. The plate was then incubated for 30 mins at 37°C, 5% CO2 in a humidified atmosphere, and cell viability was determined by measuring the resulting fluorescent signal using the Infinite M1000 PRO in FI top mode. Again, the z-positioning with integrated background correction function was used to achieve maximum sensitivity.5
The average for each dilution was calculated (based on six separate wells) and corrected by subtracting the average blank. This value was then related to the average of untreated control samples, and the respective error bars were calculated.
Results and Discussion
Incubation time study
Figure 1 and Table 1 show the results of the incubation time study using the Infinite M200 PRO and GCM. After 15 mins of incubation, the data shows good linearity and a LOD of around 80 cells per well. Incubation periods of over 30 mins increase the overall signal intensity, but do not significantly improve the LOD, making a 30 min incubation sufficient to achieve the maximum sensitivity.
The optimized incubation period of 30 mins also proved to be sufficient for cytotoxicity analysis. The dose response curve in Figure 2 shows the cytotoxic effect of STS on A431 cells. As indicated by the small error bars, the uniformity of the measurements was excellent, thanks to the high sensitivity of the Infinite M1000 PRO.
This study demonstrates the suitability of the Infinite M200 PRO and the Infinite M1000 PRO multimode readers for determination of cell viability using the PrestoBlue reagent. The dramatic decrease in incubation time compared to common resazurin-based assays was clearly verified in this study, offering increased efficiency and better assay performance.1
The Infinite M200 PRO’s incubation functions (temperature control and GCM) enable an automated assay procedure without the need for manual transfer of microplates between an incubator and the reader. In addition, the Infinite M1000 PRO’s high-end fluorescence optics were shown to be perfectly suited to detection of the fluorescent signal in the PrestoBlue assay, resulting in excellent uniformity. The use of the PrestoBlue viability reagent with the Infinite series multimode readers therefore offers a powerful solution for in vitro cell viability analysis in both assay development and screening applications.
4. Tecan Technical Note: Improved fluorescence top measurements; 396233 V.1.0, 03.2010
5. Tecan Technical Note: Maximize signal to blank intensity ratios; 396815 V.1.0, 08-2011