Estimating the number of viable animal cells in multiwell culture - A tetrazolium-based assay

Gale Haslam, Mark Richter, Diane Wyatt, Qizhuang Ye, Paul Kitos

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.

Original languageEnglish (US)
Pages (from-to)187-195
Number of pages9
JournalAnalytical Biochemistry
Volume336
Issue number2
DOIs
StatePublished - Jan 15 2005
Externally publishedYes

Fingerprint

NADP
Cell culture
Assays
Animals
Cells
Glucose-6-Phosphate
Glucosephosphate Dehydrogenase
Octoxynol
Methylphenazonium Methosulfate
Formazans
Phosphogluconate Dehydrogenase
Growth Inhibitors
Eukaryotic Cells
Insects
Cytoplasm
Color
Cell Culture Techniques
Cell Count
Cell Line
Oxidation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Estimating the number of viable animal cells in multiwell culture - A tetrazolium-based assay. / Haslam, Gale; Richter, Mark; Wyatt, Diane; Ye, Qizhuang; Kitos, Paul.

In: Analytical Biochemistry, Vol. 336, No. 2, 15.01.2005, p. 187-195.

Research output: Contribution to journalArticle

Haslam, Gale ; Richter, Mark ; Wyatt, Diane ; Ye, Qizhuang ; Kitos, Paul. / Estimating the number of viable animal cells in multiwell culture - A tetrazolium-based assay. In: Analytical Biochemistry. 2005 ; Vol. 336, No. 2. pp. 187-195.
@article{c4f62b29dfa849bd8b14d106dcc71913,
title = "Estimating the number of viable animal cells in multiwell culture - A tetrazolium-based assay",
abstract = "A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.",
author = "Gale Haslam and Mark Richter and Diane Wyatt and Qizhuang Ye and Paul Kitos",
year = "2005",
month = "1",
day = "15",
doi = "10.1016/j.ab.2004.10.008",
language = "English (US)",
volume = "336",
pages = "187--195",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Estimating the number of viable animal cells in multiwell culture - A tetrazolium-based assay

AU - Haslam, Gale

AU - Richter, Mark

AU - Wyatt, Diane

AU - Ye, Qizhuang

AU - Kitos, Paul

PY - 2005/1/15

Y1 - 2005/1/15

N2 - A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.

AB - A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.

UR - http://www.scopus.com/inward/record.url?scp=11144348940&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=11144348940&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2004.10.008

DO - 10.1016/j.ab.2004.10.008

M3 - Article

C2 - 15620883

AN - SCOPUS:11144348940

VL - 336

SP - 187

EP - 195

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -