Cell Proliferation Assays: Improved Homogeneous Methods Used to Measure the Number of Cells in Culture Terry L. Riss and Richard A. Moravec
I. I N T R O D U C T I O N
Over the last decade several improvements have been made in assay technology to enable miniaturization and more efficient measurement of the number of cells present in microwell plates. A variety of different methods have been optimized for convenient use in multiwell formats, making it easier to do large numbers of assays. The most significant improvement in efficiency has been the development of homogeneous "add, mix, and measure" assay formats compatible with robotic automation for high-throughput screening (HTS) of test compounds. Making a choice among available assay formats often depends on the preference for which marker is measured or the level of sensitivity required. Homogeneous assays are now available to measure total cell number, viable cell number, the number of dead cells present in a population, or the number of cells undergoing apoptosis. For many experimental systems the most useful information is the number of viable cells at the end of a treatment period. The parameter used most conveniently to determine the number of viable cells in culture is measurement of an indicator of active metabolism. This article describes four options for measuring cell number that are based on assaying different aspects of cellular metabolism. The example assays chosen include ATP quantitation, tetrazolium Cell Biology
reduction using [3-(4,5-dimethylthiazol-2-yl)-5-(4sulfophenyl)-2H-tetrazolium, inner salt (MTS), resazurin reduction, and total lactate dehydrogenase (LDH) activity measurement. The four examples are all homogeneous methods sensitive enough to detect cell numbers typically used in automated high-throughput 96- and 384-well plate formats. The methods are equally suitable for measuring just a few samples processed manually. In addition, all of these assays have been shown to be reproducible and exhibit good Z'-factor values (Zhang et al., 1999) desirable for automated HTS applications. Each assay has its own set of advantages and disadvantages that contribute to the decision of which one to choose.
RPMI 1640 culture medium containing 15 mM HEPES (Cat. No. R-8005), 2-mercaptoethanol (Cat. No. M-7154), trypan blue solution (0.4% Cat. No. T-8154), and phenazine ethosulfate (PES; Cat. No. P-4544) are from Sigma. Fetal bovine serum (Cat. No. SH30070) is from Hyclone. Ninety-six-well plates with opaque white walls and clear bottoms (Cat. No. 3610) are from Corning. The CellTiter-Glo luminescent cell viability assay (Cat. No. G7571) for determining ATP content, the CytoTox-ONE homogeneous membrane integrity
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CELL AND TISSUE CULTURE: ASSORTED TECHNIQUES
assay (Cat. No. G7891) for determining total LDH activity, the CellTiter 96 AQueous one solution cell proliferation assay (Cat. No. G3580) for measuring MTS tetrazolium reduction, the CellTiter-Blue cell viability assay (Cat. No. G8080) for measuring resazurin reduction, and recombinant human interleukin (IL)-6 (Cat. No. G5541) are from Promega.
0-2.0ng/ml. An equivalent volume of assay medium without IL-6 is added to sets of negative control wells. Assay plates are cultured for 72h at 37~ with 5% CO2 before processing with each of the cell number assays.
A. MTS Tetrazolium Reduction Assay
Viable cells reduce tetrazolium compounds into intensely colored formazan products that can be detected as an absorbance change with a spectrophotometer. The amount of formazan color produced is directly proportion to the number of viable cells in standard culture conditions. Cells rapidly lose the ability to reduce tetrazolium compounds shortly after death, which enables tetrazolium reduction to be used as an indicator of viable cell number. The first MTT tetrazolium...