Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay

Michael U. Shiloh, Jia Ruan, Carl Nathan

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S- nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91- kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

Original languageEnglish (US)
Pages (from-to)3193-3198
Number of pages6
JournalInfection and Immunity
Volume65
Issue number8
StatePublished - 1997

Fingerprint

Phagocytes
Fluorescence
Macrophages
Nitric Oxide Synthase Type II
Salmonella typhimurium
Survival
S-Nitrosoglutathione
Bacteria
Hydrogen Peroxide
Neutrophils
Coloring Agents
Escherichia coli
Costs and Cost Analysis
superoxide-forming enzyme
resazurin

ASJC Scopus subject areas

  • Immunology

Cite this

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay. / Shiloh, Michael U.; Ruan, Jia; Nathan, Carl.

In: Infection and Immunity, Vol. 65, No. 8, 1997, p. 3193-3198.

Research output: Contribution to journalArticle

@article{5d837764720f4632bfe1ae163c15fc09,
title = "Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay",
abstract = "To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S- nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91- kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.",
author = "Shiloh, {Michael U.} and Jia Ruan and Carl Nathan",
year = "1997",
language = "English (US)",
volume = "65",
pages = "3193--3198",
journal = "Infection and Immunity",
issn = "0019-9567",
publisher = "American Society for Microbiology",
number = "8",

}

TY - JOUR

T1 - Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay

AU - Shiloh, Michael U.

AU - Ruan, Jia

AU - Nathan, Carl

PY - 1997

Y1 - 1997

N2 - To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S- nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91- kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

AB - To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S- nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91- kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

UR - http://www.scopus.com/inward/record.url?scp=0030748692&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030748692&partnerID=8YFLogxK

M3 - Article

VL - 65

SP - 3193

EP - 3198

JO - Infection and Immunity

JF - Infection and Immunity

SN - 0019-9567

IS - 8

ER -