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Validation of a Manual Cell Counting Assay for Assessing the Kinetics of Cell Unclumping Reagents in Drug Discovery
Author(s) -
Zem Gregory,
Nahapetyan V,
Crocker K,
Tobar D,
Santos S Delos,
Nazarian D,
Hovannes J,
Beltran G,
Dubin R,
Reque L,
Singh P,
Sarkisyan L,
Cardona B,
Bachinela G,
Oppenheimer S B
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.811.8
Subject(s) - reagent , cell counting , cell , yeast , fixative , chemistry , chromatography , biology , biochemistry , cell cycle , cytoplasm
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 Information Supported 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 .