Cell-based quantitative evaluation of the MTT assay

OBJECTIVE: To analyze the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on a per cell basis and evaluate its modulation as a function of different stages of cell metabolism. STUDY DESIGN: Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area) of V79 cells and cultured macrophages were recorded for individual cells by means of digital image analysis. The effect of different serum (0-10% vol/vol) or genistein (0-100 microM) concentrations was used to modulate the MTT-specific activity response. RESULTS: As cells in culture are heterogeneous in cell size, the contribution of each cell to the total amount of formazan formed per dish is variable. The production of formazan per cell as a result of MTT bioreduction was found to be proportional to cell size. CONCLUSION: Specific MTT-reducing activity was analyzed in phagocytes and nonphagocyte cells, revealing the utility of this variable in evaluating the MTT assay at the single-cell level.     

A quantitative assay for aggrecanase activity.

Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N-terminal ARGSVIL sequence and a second anti-aggrecan peptide antibody. For higher sensitivity of the assay, P1-P5 residues of the aggrecanase site within the aggrecan substrate were changed by in vitro mutagenesis. Specific activities of recombinant truncated ADAMTS1 and ADAMTS4 estimated with authentic aggrecan interglobular domain amounted to 2.4 +/- 0.4 and 21.7 +/- 9.5 nmoles hydrolyzed substrate/min.mg, respectively. The values were 10.3 +/- 5.1 and 151.5 +/- 93.5 nmoles/min.mg for hydrolysis of the modified substrate. The aggrecanase activity assay can be used for (1) kinetic characterization of aggrecanase activities of human and animal ADAMTS, (2) screening of inhibitors for aggrecan hydrolyzing ADAMTS, and (3) estimation of aggrecanase activities in biological samples.     

A simple assay for the quantitative evaluation of Phospholipase D activity

Summary We describe the preparation of phosphatidyl p-nitrophenol (PpNP) and its use as a substrate in an assay for the quantitative evaluation of Phospholipase D. The solution of the substrate in Tris HCl buffer, pH 8, and Triton X is mixed directly with an aliquot of fermentation broth and the absorbance of the solution read at 405 nm. The method is rapid, sensitive and inexpensive.     

A quantitative assay for the juvenile hormones and their precursors using fluorescent tags

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.

Methods

A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).

Conclusions

The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.

Significance

The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.     

Protein kinase assay on isoelectric focusing gels: evaluation of its quantitative potentials.

The conditions of the Hirsch-Rosen assay (1974, Anal. Biochem.60, 389–394) for protein kinase activity oncomplete isoelectric focusing gels have been analyzed and further developed with the consequence that the test can be easily adapted to other protein kinases in a quantitative manner. Special attention was given (i) to the breakdown of the pH gradient in relation to gel size and Ampholine concentration in order to achieve optimal pH ranges for the assay, (ii) to the introduction of histone as a substrate besides protamine, and (iii) to the distribution of the particular substrates and products throughout the gel. The results of the protein kinase assay on gels were shown to be linear for at least 1 h, and to be dependent on the amount of ATP and on the amount of protein kinase applied, thereby fulfilling the requirements necessary to yield quantitative data.     

A quantitative assay for fragmented DNA in apoptotic cells.

Cleavage of cellular chromatin at internucleosomal sites is a characteristic change of DNA integrity in cells undergoing apoptosis. We have developed an assay for quantitation of internucleosomal DNA fragmentation in apoptotic cells. This technique involves purification of cellular DNA, dephosphorylation of the DNA ends, labeling of DNA with 32P at the 5'-end, gel electrophoresis through agarose, and quantitation of the radioactivity in DNA bands. This assay, which is about 1000- to 2000-fold more sensitive than visualization of DNA bands by ethidium staining, allows the detection of DNA fragments at picogram levels. A method for quantitatively determining the number of fragmented DNA strands is also described. Application of this new assay to evaluate the time course of internucleosomal DNA fragmentation was demonstrated in apoptotic cells induced by an anticancer nucleoside analogue.     

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.     

Enantioselective electrochemical oxidation of enol acetates using a chiral supporting electrolyte

Anodic oxidation of 1-acetoxy-3,4-dihydronaphthalene (1) and alpha-acetoxy-beta-alkylstyrenes (3) at -78 degrees C in a mixed solvent of acetonitrile (CH(3)CN), tetrahydrofuran (THF), and acetic acid (AcOH) containing (S)-tetraethylammonium camphorsulfonate as a chiral supporting electrolyte brought about enantioselective formation of the corresponding 2-acetoxy-1-tetralones (2) and (R)-2-acetoxy-1-phenyl-1-alkanone (4) with maximum enantiomeric excess (ee) of 44% and 21%, respectively. Introduction of a 7-methoxy group into 1 and increase in bulkiness of a beta-alkyl group in 3 resulted in improvement of enantioselectivity of the reactions.     

Enzymic method for the quantitative determination of reduced glutathione

A new specific and sensitive assay method for reduced glutathione has been developed. It is based on the reaction HCHO + NAD⁺ + H₂O → HCOOH + NADH + H⁺, catalyzed by formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC.1.2.1.1) in the presence of reduced glutathione. Oxidized glutathione and other thiols do not interfere in this reaction. A purification procedure for formaldehyde dehydrogenase from beef liver is presented.The influence of cysteine and some other thiols, leucine, ascorbate, lactate, pyruvate and four acids generally used for deproteinization is reported. The results obtained by this method from human blood and rat tissues are compared with those obtained by Ellman's method.     

A quantitative assay for collagen synthesis in microwell fibroblast cultures.

A new method for estimating collagen synthesis in microwell cultures of fibroblasts is presented, ³H-Labeled-proline-collagen was purified by successive salt precipitations at acid and neutral pH in the presence of carrier collagen. Variability between replicates was less than 10% (standard deviation) and recovery of labeled collage internal standards was greater than 90%. More than 90% of recoverable radioactivity was in collagen as demonstrated by polyacrylamide gel electrophoresis and carboxymethyl cellulose chromatography. The culture system is highly reproducible and allows use of a large number of separate cultures with uniformity of culture conditions and economy of reagents.