engleski

Non-radioactive fluorescence-based cell quantitation assay development

A cell count/quantitation assay has been proven by High Content Screening research to be the most valuable assay for determining drug toxicity (i.e. cyto-static or cyto-toxic effects). A cell quantitation assay can indicate drug toxicity by multiple mechanisms, so its therapeutic application areas are very broad. Radioactive cell proliferation assays (i.e. thymidine incorporation) are precise, but have potential risks. They are tedious and do not label live cells stopped in the cell cycle and they are not designed to measure drug cyto-toxicity (decreases in cell numbers below control values). The most reliable cell quantitation assay uniformly labels all live cells, quiescent or dividing, preferably with a sensitive and safe fluorescence method. Simple cell counting by hemocytometer is the most reliable method of cell quantitation, but it is tedious and not adaptable to HTS automation. The calcein AM cell labelling dye is the premier indicator of cell quantitation techniques. Calcein AM is non-fluorescent until cleaved by intracellular esterases only in live cells to the product calcein. Calcein has six negative charges, so it is excellently retained by cells for hours. Our results show that calcein fluorescence has an excellent correlation to live cell number (r=0.997) for peripheral blood mono-nuclear cells (PBMCs), in the range of 0-100000 cells/well and the calcein method can easily be adapted to HTS automation in the 96-well plate format. Using 2.5 μM levels, the calcein lower detection limit is 800 PBMC/well and the signal-to-noise ratio is as high as 16:1. The calcein assay produces well-defined dose response curves with the classical sigmoidal shape. The calcein cell quantitation assay can measure both decreases in cell number (i.e. cyto-toxicity) and increases in cell numbers (i.e. proliferation), relative to the control wells. The cost of the calcein method is about one-fourth the price of the 3H-thymidine incorporation assay or cyto-toxicity kits requiring dye metabolism (i.e. the Alamar Blue assay). Our results reveal that the calcein assay is a reliable method for cell quantitation, useful in drug evaluations.

calcein; fluorescence; high content screening; PBMC

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