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Edge assay: kinetic analysis of reagents affecting cell clumping
Author(s) -
Zem Gregory,
TerPapyan H,
Arvizu S,
Nernsuan J,
Rabizadeh S,
Amidi M,
Nazari S,
Waas S,
Lee S,
Orujyan A,
Manookian L,
Taghinia P,
Carpio C,
Trinh D,
Balazadeh H,
Oppenheimer S B
Publication year - 2013
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.27.1_supplement.650.14
Subject(s) - yeast , reagent , distilled water , serial dilution , chromatography , saccharomyces cerevisiae , chemistry , biology , biophysics , biochemistry , medicine , pathology , alternative medicine
Cells in clumps are often more dangerous than single cells in a cancer setting and in infectivity. We present a fine‐tuned method to test reagents that may affect cell clumping. In each experiment two 0.2ml droplets of distilled water were placed on glass microscope slides to which 1% formaldehyde fixed yeast (Saccharomyces cerevisiae) in distilled water (pH adjusted to 4.0) were added at dilutions that resulted in 8–25 single yeast and clumped yeast cells that were observed at the edge of the droplets, providing a standardized reference point for counting single vs clumped (two or more) yeast. Using birchwood toothpicks, various concentrations of solid reagents (salts, sugars, complex carbohydrates, amino acids) (1–10 mg) were added to the experimental drops and nothing was added to the control drops. Numbers of single and clumped yeast were counted before addition of reagents, immediately after addition and at 20, 40, 60 min after reagent additions in 1179 trials (21 C). The numbers of single yeast and yeast clumps were observed with a light microscope (100–200 X) and recorded at each step. The percentages of single yeast in experimentals and controls were plotted showing SEM error bars and t‐tests were performed at 20,40,60 min comparing the percentage of single yeast in experimentals vs controls. P values of less that 0.05 suggested that the percentages of single cells in the experimentals were significantly different than in the controls. The results show that the precise analysis that this assay affords is able to detect very small yet statistically significant differences in reagent effects and could lead to identification of reagents that may be further developed into anti‐clumping drugs (Supported by NSF Presidential Award 0731633, NIH SCORE, RISE, MARC, the Joseph Drown Foundation, the Sidney Stern Memorial Trust).