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Purified high molecular weight synthetic A?(1-42) and biological A? oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

ABSTRACT: Synthetic soluble A? oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of A? oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10-100 nM). Purified oligomers, but not monomers or fibrils, elicited an increase of a detergent-insoluble form of MTT formazan within 2h as opposed to a control toxin (H(2)O(2)). This effect was comparable for biological and synthetic peptide in both cell types. Monomeric A? attenuated the effect of soluble oligomers. This study suggests that the activities of biological and synthetic oligomers are indistinguishable during early stages of A? oligomer-cell interaction.

SUBMITTER: Weidner AM

PROVIDER: S-EPMC3110692 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Purified high molecular weight synthetic Aβ(1-42) and biological Aβ oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

Weidner Adam M AM Housley Molly M Murphy M Paul MP Levine Harry H

Neuroscience letters 20110409 1

Synthetic soluble Aβ oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of Aβ oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10-100 nM). Purified oligomers, but not monomers ...[more]

PMID: 21504780

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Project description:The MTT assay, based on the enzymatic reduction of the water-soluble, yellowish tetrazolium salt 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) to purple formazan, is commonly used for assessment of cell viability and proliferation. Accurate performance by the MTT assay depends on complete solubilization of cells and formazan and stability of the colored solution. Comparison of different solubilization solutions revealed that dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), buffered with ammonia buffer, pH 10, and containing 5% SDS, produced the best results. These two solvents provided rapid and complete solubilization of formazan and cells, with minimal background absorbance at 700 nm, good reproducibility (low interassay coefficient of variation), high sensitivity, and color stability for at least 24 h. A linear relationship between viable-cell number and formazan absorbance was preserved for cell densities up to ∼1 × 109 cells/mL for Gram-negative and Gram-positive microorganisms. Since MTT can be reduced by medium components in the absence of cells, blanks containing all medium components but no cells should be run simultaneously. Measurements at two wavelengths, one corresponding to absorption peak of formazan (570 nm) and a background absorbance far from the peak (700 nm), are necessary to avoid artifacts due to incomplete solubilization and turbidity. IMPORTANCE Reduction of the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol)-2,5 diphenyl-tetrazolium bromide (MTT) to purple, water-insoluble formazan is commonly used for assessment of cell viability and proliferation. Spectrophotometric detection of formazan requires its solubilization. The solubilization solvent has a strong influence on data acquisition and often introduces artifacts, leading to misreading of results. This study offers a choice of solvents that minimize solubilization artifacts when the MTT test is applied to microbiological cultures.

Dataset Information

Purified high molecular weight synthetic A?(1-42) and biological A? oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

Publications

Purified high molecular weight synthetic Aβ(1-42) and biological Aβ oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

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