A kinetic assay for non‐automated drug screening

Abstract

A non‐automated kinetic assay has been used in this laboratory to screen reagents that unclump fixed yeast, a model for unclumping human cancer cells, human blood cells and pathogens (FASEB J, 26:62ab; 28:234ab; 29:65ab; 31:539ab). Fixed yeast is the material of choice because its surface properties are similar to live cells without unknowns associated with live cell metabolism (Acta Histochem 104: 99–106). Here the assay is tested to determine if it also can be used to assess cell clumping instead of unclumping, of importance in many drug screening venues, using a calcium induced flocculation model. In addition, the precision of the assay is tested using reagents that do not clump or unclump cells. O.05 ml of Prefer (Anatech Ltd, Battle Creek, MI) fixed yeast (Saccharomyces cerevisae) were added to 1 ml droplets of deionized water on glass microscope slides. The percentages of single yeast and clumps (2 or more cells) were assessed with light microscopes (100X, 200X) before and after addition of 1, 2 or 3 mg of calcium sulfate dihydrate (droplets with no additions served as controls) and at 20 min, 40 min and 60 min with agitation. The results showed that calcium sulfate flocculated the yeast in a concentration and time dependent manner. For example for calcium sulfate, 1, 2 and 3 mg per ml, in 30 trials, with p values generally below 0.05 and tight standard error bars, the percentages of single yeast at 60 min were: 1 mg/ml 40 percent vs 67 percent in controls, 2 mg/ml 27% vs 67 percent in controls, 3 mg/ml 14 percent vs 72 percent in controls. The assay, therefore, assesses the kinetics of cell clumping (flocculation). When 3 reagents, D‐melizitose, D‐melibiose, L‐ascorbic acid were tested at 1, 2 and 3 mg per ml at 20, 40 and 60 min, the assay picked up little or no effect on the yeast. For example in 90 trials with tight error bars each time point showed little to no significant difference between experimentals and controls. The results indicate that the assay kinetically measures cell clumping and is reproducible and precise in generating data. The non‐automated assay is ideal for drug screening by hundreds of investigators at a time especially when expense is a consideration.Support or Funding InformationCSUN Foundation and Corporation Accounts, U.S. Presidential AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.