An improved scintillation cocktail of high-solubilizing power

A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for ³H (47%) and ¹⁴C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon.     

Improvements in and Environmental Applications of Double-Vial Radiorespirometry for the Study of Microbial Mineralization

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with ¹⁴C-labeled glucose, acetate, and glutamate and [¹⁴C]cellulose- and [¹⁴C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.     

Tritosol: A new scintillation cocktail based on Triton X-100

A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for³H are about 47%, for¹⁴C about 87%, and for⁴⁵Ca about 80%.     

Liquid scintillation counting of polyacrylamide gels crosslinked with N,N′-methylene-bis-acrylamide and N,N′-diallyltartardiamide

Tritium (³H)-labeled material within polyacrylamide gel slices is commonly quantified by four general liquid scintillation counting methods: combustion, gel solubilization, selective solubilization of modified crosslinked gels, and elution. Of these four methods examined in this study, only combustion ensured complete recovery of ³H label; however, a less expensive and more convenient elution method yielding both a high recovery and cocktail counting efficiency was the use of Soluene-350 with 0.55% Permablend III in toluene. This particular solubilizer-cocktail system eliminates almost all chemiluminescence as does combustion.     

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.     

A convenient, efficient, and inexpensive method for scintillation counting of polyacrylamide gel slices after electrophoresis

A method is described for analyzing the radioactivity of ³H-labeled RNA after separation by gel electrophoresis. The gels were soaked in 10% acetic acid and were sliced. The gel slices were dehydrated in alcohol, then saturated with toluene, and finally permeated with a toluene-based scintillation fluid containing butyl-PBD. The radioactivity of RNA was then analyzed in situ in the gel slices using a liquid scintillator. The counting efficiency of this technique is high, about 58%. This is even slightly better than the counting efficiency attained after solubilization of the RNA in Soluene 350 (about 55%). With the same fluor, Permablend III, the counting efficiencies of the two techniques are alike.     

Measurement of 131 I and 125 I by liquid scintillation counting

When aqueous samples are made miscible with a toluene scintillation solution by means of Bio-Solv BBS-3, high ¹²⁵I and ¹³¹I efficiencies can be achieved over a considerable range of “impurity” quenching, and adequate isotope separations can be achieved using liquid scintillation counters with “linear,” “pseudologarithmic,” or “logarithmic” amplification. Using an example of each sort of counter, we have graphically outlined the two somewhat different procedures for choosing the best instrument settings for single- and double-isotope counting. “Linear” instruments, despite a slightly more complex procedure for selection of settings, may offer the advantage, in double-isotope counting, of somewhat greater isotope separations because of the greater attenuation of photo-electron spectra.     

A water-miscible, nonhazardous liquid scintillation cocktail

The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts ¹⁴C with 80% efficiency and ³H with 17% efficiency. It has a high flash point (over 300°F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications.     

Preparation of 14C-labeled algal samples for liquid scintillation counting

Four commonly used techniques for preparation of ¹⁴C-labeled algal samples on membranes for liquid scintillation counting were compared and a simple technique for apparent net assimilation measurement from aqueous samples was introduced. All four techniques yielded similar radioactivities from the test cultures and are thus suitable for measurements of ¹⁴C algal samples. The possibly carcinogenic solvent dioxane was not necessary with PCS scintillation cocktail for dissolving radioactivity from algae on filters.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

A thermostable cocktail for the liquid scintillation counting of heterogeneous media

Considerable analytical errors arise in the liquid scintillation counting of heterogeneous media as a consequence of gel instability. With large sample numbers, a major causative factor of this instability is temperature changes during the counting period. An emulsifier-scintillation cocktail has been designed to provide stable counting conditions for heterogeneous media over a temperature range of 10–30°C, i.e., the wide range of temperature likely to be encountered in liquid scintillation counters lacking sample cooling facilities. A comparison was made with a conventional commercially available emulsifier-scintillator.     

The identification of membrane glycocomponents in polyacrylamide gels: A rapid method using I-labeled lectins

A rapid method is described for the identification of lectin binding membrane glycocomponents in polyacrylamide gels. The method requires only small quantities of membrane material and is applicable to a wide variety of lectins. Solubilized membrane components are electrophoretically separated according to molecular weight in a SDS-polyacrylamide gel. The gel is then incubated in a solution containing the ¹²⁵I-lectin. After elution of unbound ¹²⁵I-lectin, the gel is dried down and those bands which have bound the ¹²⁵I-lectin are identified by radioautography. The amount of bound ¹²⁵I-lectin can be quantified by either densitometric scanning of the radioautogram or by liquid scintillation counting of the dried gel.     

A simple method for the liquid scintillation counting of precipitated protein from red blood cells

A method for the liquid scintillation counting of precipitated protein from red cells in 0.1–1.0 ml of blood is described. Precipitate is incubated for 0.5 hr at 100°C with equal volumes of acetic acid, ethyl acetate, and hydrogen peroxide; an equal volume of hydrochlorie acid is then added, followed by a toluene/Triton X-100 scintillation mixture containing primary and secondary scintillators. Maximum counting efficiencies with precipitate from 0.2 ml of blood were 90% for ¹⁴C and 35% for ³H. Recovery of labeled amino acid was not less than 90%. Chemiluminescence decayed to not more than 15 cpm above background in 45 min.     

Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Counting efficiency in the radioassay of 3H- and 14C-labeled lipids adsorbed on silica gel by liquid scintillation

The effect of silica gel on the recovery of radioactivity from ¹⁴C- and ³H-labeled lipids by liquid scintillation is analyzed. In the most unfavorable ease, when counting with a toluenefluor solution directly added to the vials containing the adserbed lipid, drastic reductions in the counting efficiency were found, which depended on the amount of silica gel, sample activity, and chemical nature of the lipid. For certain lipids like phosphatidylcholine, these effects were not completely overcome even by introducing water and detergents in the counting system. This paper intends to draw attention to the fact that these factors should be especially taken into account when comparing different lipids from thin-layer chromatograms.     

Radiorespirometry and metabolism of cultured cells attached to petri dishes

A radiorespirometer was constructed for continuous quantitation of ¹⁴CO₂ released from specifically labeled substrates by intact cultured cells attached to plastic petri dishes. An airtight chamber is created by sealing the petri dish with a specially designed cover inside a thermostated holder. Rapid equilibration of released ¹⁴CO₂ with a 5% $\text{CO}{}_2\text{95\%}$ air carrier gas is achieved by bubbling the carrier gas under the surface of the growth medium. Labeled CO₂ is removed from the carrier gas by trapping in an organic base and quantitated by liquid scintillation counting. Additions to or sampling of the growth medium may be performed during a run and the carrier gas may be modified to test the effects of anesthetics and different O₂ levels. The ability to continuously monitor ¹⁴CO₂ release can provide valuable information concerning the metabolic pathways of substrate oxidation which cannot be obtained from single ¹⁴CO₂ determinations. A capacity of 12 culture plates enormously increases the amount of data that can be collected in a given time. The use of liquid scintillation counting increases the sensitivity and resolution over the ion chamber and Geiger counter methods, and permits utilization of the procedure in a much wider range of laboratories. Data obtained for the oxidation of specifically ¹⁴C-labeled glucose and pyruvate by neonatal rat heart cells in culture, in both the presence and absence of oxygen, are provided as examples of the utility of the method.     

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.     

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.     

Biosynthesis of the sesquiterpenoid,capsidiol, in sweet pepper fruits inoculated with fungal spores

Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2-¹⁴C] or RS-mevalonolactone-[2-¹⁴C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2-¹³C₂] into capsidiol, as indicated by the pattern of ¹³C-¹³C coupling from ¹³C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.     

An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).