Liquid scintillation counting of radioactive monomeric and polymeric carbohydrates on paper chromatograms

A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol.The water elution-Aquasol procedure allows water-soluble substances (¹⁴C- and ³H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.     

Microdetermination of inorganic sulfate using thin-layer plates

Inorganic sulfate was precipitated on cellulose thin-layer plates with the radioactive reagent, ¹³³BaCl₂. Excess reagent was removed by repeated washings with an acidic BaCl₂ solution. The residual activity was transferred to vials by cutting out the point of application and its immediate surroundings. Counting was performed in a scintillation well γ-counting system. The concentration-activity curve was linear.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.     

Manometric determination of CO2 combined with scintillation counting of C-14

A procedure is described for rapid transfer to scintillation vials of ¹⁴CO₂ evolved by reactions in the mechanically shaken manometric chamber of Van Slyke and Neill. Results are given with amounts of CO₂ ranging from 2 to 1000 μmoles, and with Hyamine and phenylethylamine to absorb CO₂ in the vials.     

Branched-chain-amino-acid aminotransferase assay using radioisotopes

A method for the assay of the activity of branched-chain-amino-acid aminotransferase from Escherichia coli has been developed. Radioactive isoleucine is used, the radioactive oxoacid formed in the enzymic reaction is converted to its p-nitrophenylhydrazone, and the hydrazone is extracted into toluene based scintillation liquid. The small reaction tubes containing the toluene layer and the reaction mixture as a water layer are placed into liquid scintillation vials and counted for radioactivity. The radioactive amino acid remaining in the water layer causes only a rather low background.     

A device for comparing callus growth rates in vitro

A device to compare the kinetics of callus growth in vitro is described. Changes in volumes of callus grown in scintillation vials were monitored photometrically without removing the sample from the solid support and medium. It is shown that a fiberglass-paper solid support is superior to a plastic foam solid support for the growth of American chestnut callus.     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Protection of histones isolated from elastase-treated mouse epidermis

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Problem in liquid scintillation counting: adsorption of (14-C)pholyethylene glycol in dilute solutions.

[¹⁴C]polyethylene glycol is the method of choice for quantitating changes in intestinal water flux during drug absorption experiments in animals and man. This study points out some of the problems which can be encountered in using this method and provides ways to minimize these problems. Polyethylene glycol selectively binds to the glass wall of scintillation vials during counting and results in a decrease in counting efficiency as a function of time. The results obtained when using this method are determined by the choice of scintillation vial, scintillation cocktail, concentration of polyethylene glycol and the time period over which the samples are counted.     

Liquid scintillation counting of energetic beta-emitting isotopes on solid supports: problems arising from particle range

If emitters of energetic β-particles (such as ³²P) are counted on solid supports in liquid scintillation systems, considerable distortion of the apparent energy spectrum of the β-particles can result if the material is placed on the bottoms or against the walls of vials. This results if the path length in the scintillation fluid available to a substantial proportion of the β-particles is short compared with their range. The phenomenon leads to reduced and variable efficiency in normal counting channels and to difficulties in double-labeling.     

A rapid method to determine the mammalian cell cycle

A method was developed for determining the duration of the mammalian cell cycle and each of its major phases, mitosis, G1, DNA synthetic period, and G2. Mitotic time was determined by assessment of the mitotic index at intervals after cells collected in mitosis and stored at 4 °C were reincubated at 37 °C. The duration of the three remaining phases was derived from a graphic representation of the uptake of ³H-thymidine by a synchronous population of cells grown directly in scintillation vials. The scintillation counting method for determination of these parameters is advantageous over methods using autoradiography in that the investigator's bias in scoring cells is eliminated. Complex mathematical interpretations are unnecessary, and the data obtained from the scintillation counter are readily processed. Results from scintillation counting and autoradiographic methods are shown to be comparable.     

Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis.

HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented.     

A simple apparatus for the continuous monitoring of 14CO2 production from several small reaction mixtures

A simple apparatus is described which permits the continuous monitoring of ¹⁴CO₂ production from ten separate reaction mixtures simultaneously. The device is relatively simple and inexpensive to construct, makes use of small disposable incubation vials, and allows complete trapping of all ¹⁴CO₂ evolved in scintillation vials, where it can be easily counted. The use of this apparatus to determine the rates of metabolism by glomeruli of ¹⁴C-labeled substrates to ¹⁴CO₂ is described.     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

An intact assay for enzymes that labilize C--H bonds.

The activity of enzymes that labilize known CH bonds can be estimated in intact tissue by following the loss to water of tritium from specifically labeled substrates. The accumulated tritium oxide (³HHO) in the medium is recovered by direct sublimation of aliquots of the medium into scintillation vials and the radio-activity measured by liquid scintillation spectrometry.     

A simple micro-procedure for the estimation of tritiated water in biological samples

• 1.A simple micro-procedure for the estimation of tritiated water in biological samples is described.
• 2.2. Tritiated water is quantitatively recovered from perchloric acid extracts of tissue directly into scintillation vials by elution from micro-columns of borate resin.

     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis

Summary HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented. This research was supported by Oklahoma Agricultural Experiment Station Project 1534. This publication is Article J-3334 of the Oklahoma Agricultural Experimental Station.