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This laboratory has developed and used a manual (non‐automated) cell counting assay for examining the kinetics of cell interactions in a yeast model (FASEB J 32:113; 31: 539; 30: 44; 30:49; 29: 52; 29: 65; Acta Histochem 116: 1514; 108: 311). Manual cell counting is often considered as a gold standard in some applications because of direct observation of results (Comparison of Cell Counting Methods…Inhal Toxicol 28(9): 410–429). For many years this assay has been used to evaluate the efficacy of reagents to unclump yeast cell clumps, with possible human applications in unclumping cancer cell clumps, unclumping thrombocytic blockages and reducing biofilms. In this study the assay is further validated by not only counting single cells as done in the past, but also counting cell clumps and number of cells per clump. To accomplish this, in over 400 trials by 14 investigators, sodium citrate dihydrate and magnesium sulfate were tested at 1, 2 and 3 mg per ml deionized water for their effects on fixed (Prefer fixative, Anatech Ltd, Battle Creek, MI) yeast (Saccharomyces cerevisae) over 60 min (with agitation every 20 min) on glass microscope slides and plotting all data with standard error and calculating p values comparing experimentals and controls in each category of results. It was generally observed that when there was no significant change in percentage of single cells in experimentals and controls (no reagent) there was no significant change in number of clumps and number of cells per clump. When there was a significant increase in percent single cells (suggesting that the reagent caused cell unclumping), there was a reduction in clump number and number of cells per clump. A specific example of this finding in 60 trials was that 3 mg sodium citrate dihydrate per ml deionized water caused a 35% increase in single cells by 60 min. The number of clumps decreased by 56% and the number of cells per clump decreased by 39%. Trends for magnesium sulfate were generally similar to those for sodium citrate dihydrate. In summary, this manual assay was further validated here by assessing cell clumps as well as the usual single cells. This assay is considered to be a gold standard for precise assessment of cell unclumping reagents in drug discovery.Support or Funding InformationSupported by California State University, Northridge Biology Department and Center for Cancer and Developmental Biology Foundation and Corporation accounts including U.S. Presidential Award contribution.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.