An improved method for the collagen gel contraction assay

The collagen gel contraction (CGC) assay is used frequently to study the cell-mediated reorganization of the extracellular natrix. In a typical CGC assay, cells embedded in a disk-shaped lattice (gel) of native type I collagen fibers compress the fibers and, consequently, reduce the diameter of the collagen disk within h or d. The degree to which the collagen is contracted is usually quantified by measurement of the diameter or the area of the disk. During CCC assays, friction or adhesion (or both) between gels and their culture containers can cause gels to be incompletely contracted or to acquire distorted shapes. Such occurrences degrade the reproducibility and reliability of measurements of gel dimensions. To address these problems, we developed an oil-supported collagen retraction (OSCR) assay that creates an environment of low friction and adhesion around the contracting collagen gel. The OSCR assay is accomplished with simple equipment and is easily performed, sensitive, and consistently yields fully contracted gels with minimal distortion.     

An improved method for assay of cholesterol 7 alpha-hydroxylase activity

An improved assay method for cholesterol 7 alpha-hydroxylase which is accurate, sensitive, and yet still simple is described. The method consists of two parts: the first part is cholesterol 7 alpha-hydroxylation in liver microsomes utilizing cholesterol in situ as the substrate, and the second part is conversion of the product, 7 alpha-hydroxycholesterol, into 7 alpha-hydroxy-4-cholesten-3-one having an intense absorption at 240 nm by the action of cholesterol oxidase. The converted sterol is then analyzed by high-performance liquid chromatography. During the second enzyme reaction, the first enzyme reaction is halted and further metabolism of the product is prevented. In consequence, the method had more than 10-fold increase in the sensitivity than the previous one.     

An improved clearing method for GUS assay inArabidopsis endosperm and seeds

Precise cellular localization of the GUS stain is notoriously difficult inArabidopsis seeds. Here we report an improved protocol for the clearing of seeds after GUS staining. Incubation in ethanol-acetic acid (EtAc) and Hoyer’s medium allows reliable cellular localization of the GUS, even in seeds from late developmental stages. This method also leads to the staining of nucleoli in the endosperm and embryo, facilitating nuclear counts in endosperm development. An erratum to this article is available at .     

An improved fluorometric assay for DNA

A modification of the fluorometric assay of Kissane and Robins is described. The modified procedure is very accurate, widely applicable, and reasonably easy to use. A standard cell type instead of a standard DNA solution makes the assay universally reproducible. DNA content is calculated by a simple method from the slope of a DNA concentration series. This can be used to detect technique erros.     

An improved automated method for the estimation of sialic acid released in the neuraminidase assay

An improved automated method for the estimation of sialic acid released from macromolecular substrates in the neuraminidase assay is described. The problems of introducing a dialyser into the system are outlined, and the possible causes of an apparently bimodal distribution of results are discussed.     

An improved procedure for the assay of pyruvate dehydrogenase

A modified procedure for the determination of the decarboxylation of [1-¹⁴C]pyruvate by pyruvate dehydrogenase is described which requires only small amounts of bovine kidney pyruvate dehydrogenase complex. The activity is greatly increased by the addition of high concentrations of ferricyanide.     

An improved glucoseoxidase--peroxidase-coupled assay for -fructofuranosidase activity

An improved glucoseoxidase-peroxidase-coupled assay for the determination of β-fructofuranosidase activity is described. The method makes use of the double effect of Tris (2-amino-2-hydroxymethylpropane-1,3-diol) as an inhibitor of both invertase and contaminating glucosidases. The method is very sensitive and is suitable for routine determinations. The total time needed for a single analysis is less than half an hour.     

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.     

An improved cycling assay for nicotinamide adenine dinucleotide

A new cycling assay for NAD that uses thiazolyl blue as a terminal electron acceptor has been found to offer significant advantages over the more established procedure that employs 2,6-dichlorophenolindophenol. With thiazolyl blue, the cycling assay is linear with NAD at picomole levels, and with time for at least 120 min. In contrast, with 2,6-dichlorophenolindophenol as terminal acceptor, the cycling assay deviates considerably from linearity at picomole levels of NAD, and the reaction rates become linear for shorter periods of time as the level of NAD increases. Data are given which provide a basis for choosing optimal assay conditions using the new thiazolyl blue cycling technique.     

An improved viscosimetric assay for vertebrate collagenase activity

An improved viscosimetric assay for vertebrate collagenase acitivity is described. The assay is carried out at 35 degrees C in the presence of 1 M glucose to prevent fibril formation. The decrease in viscosity is linear with ime and proportional to enzyme concentration.     

An improved protein binding assay for cyclic AMP

A simplified and inexpensive protein binding assay for cAMP has been developed for the rapid measurement of subpicomole quantities of the nucleotide. Conditions of assay were established in which interferences from salts and nucleotides present in biological samples were eliminated. Sodium chloride was demonstrated to reduce cAMP binding by denaturing the regulatory subunit of the protein kinase.     

An improved resazurin-based cytotoxicity assay for hepatic cells

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye. This revised version was published online in July 2006 with corrections to the Cover Date.     

An improved method for the synthesis of flavanones

An isomerization of 2'-hydroxychalcones into the corresponding flavanones in ethanol in the presence of triethylamine is described.