A simple program which processes liquid scintillation counting data from samples containing both 125I and 3H under conditions of varying quench

A simple program was written for a programmable calculator which enabled simultaneous measurement of ³H and ¹²⁵I by liquid scintillation counting. The program uses quench correction curves to calculate the effects of quench in each sample and calculates the amount of each isotope present by solving a linear equation set simultaneously.     

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated duringin vivoexpression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of³H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.     

Counting emissions from potassium-42 in plant tissue with a liquid scintillation spectrometer

Summary A method has been devised to count emissions from K⁴² in plant tissue by liquid scintillation spectrometer with low quenching. The plant samples were ashed in aluminium dishes, folded into aluminium wafers and suspended in the liquid scintillator of counting vials. The samples were counted in a scintillation spectrometer at one to five minute intervals seven or more times and until the total exceeded 1000 counts. The efficiency of counting was approximately 70%.A computer program was written to calculate decay corrections for the short half-life isotope K⁴². The program computed the weighted mean of the counts, standard deviation of the mean, percentage standard deviation of the mean and Chi-square value for each sample. The measurements were precise enough for K-uptake studies.     

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%.     

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.     

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.     

Technique for Measuring 14CO2 Uptake by Soil Microorganisms In Situ

Uptake of ¹⁴CO₂ in soils due to algae or sulfur-oxidizing bacteria was examined by incubation of soil samples with gaseous ¹⁴CO₂ and subsequent chemical oxidation of biologically fixed radioactive isotope to ¹⁴CO₂ for detection with a liquid scintillation counting system. The ¹⁴CO₂ was added to the soil in the gas phase so that no alteration of the moisture or ionic strength of the soil occurred. Wet oxidation of radioactive organic matter was carried out in sealed ampoules, and the ¹⁴CO₂ produced was transferred to a phenethylamine-liquid scintillation counting system with a simply constructed apparatus. The technique is inexpensive and efficient and does not require elaborate traps since several possible interfering factors were found to have no harmful effects. Experiments in coal mine regions and in geothermal habitats have demonstrated the ecological applicability of this technique for measurement of CO₂ fixation by sulfur-oxidizing bacteria and soil algae.     

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.     

Genotypes Analyses of Ten Genes Related to Eating Quality of Twenty-three Fragrant Rice Cultivars by Molecular Marker

Fragrance is a very important quality traits of rice, fragrant rice research has been an important research issue in rice research fields in nowadays. In our study, using the detection method of molecular markers, genotypes of the 10 genes (Wx, SSII 3, SBE3, AGPiso, SSIII 2, AGPlar, PUL, SSI, ISA, SSIV 2) associated rice eating quality were analyzed in the 23 fragrant rice cultivars. The result indicated that the “2845”rice cultivar possesses the best combination of genotypes related to the highest eating quality, and the next best rice cultivars were “Songxiangzaojing”, ”Suhuxiangjing”, “B1”, “Wuyun2645”, “Tongyunjing”, “Yinxiang28”, “Xiangjing49②”, “99983”, “Wxiang99075”, “07-08”, “Yunjingyou15”, “29185”, “Nanhai318”. Other 9 rice cultivars (“Dahuaxiangjing”, “Wuxiang14”, “Xiangjing”, “Della”, “Dalixiang”, “Taiguoxiangdao”, “Cxiang517”, “Xiangdao No1”, “Zhongxiang No1”) obtain some of bad genotypes which may have adverse effect on rice eating quality. This study was helpful to establish an overall understanding of the genotypes of these ten eating quality related genes in the 23 tested fragrant rice cultivars, and provides the important genotype information in selecting and cultivating new fragrant rice with good eating quality in future via molecular markers assisted selection method.     

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.     

The determination of the radioactivity of 14C samples adsorbed on glass vials by direct liquid scintillation counting

A method for obtaining the true activity and counting efficiency of a ¹⁴C sample partially or completely adsorbed on the walls of a counting vial by liquid scintillation counting is presented.     

Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.     

Increased scintillation counting of 3H-amino acids bound to transfer RNA.

Approximately a threefold higher tritium scintillation counting efficiency was observed for the ³H-labeled amino acids covalently bound to tRNA, compared to the nonbound ³H-amino acids used as counting efficiency standards.     

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.     

Structural studies of hemoglobin chains from Virginia white-tailed deer

1.

1. Structural and quantitative studies have been made on hemoglobins of Virginia white-tailed deer, Odocoilus virginianus, with emphasis on the α chains. Hemoglobins with ^Iα chains have been termed “major” and those with ^IIα chains “minor.” Previous studies of over 400 deer have shown that all animals produce major components, but many do not have the minor components. These observations as well as data showing that the minor component can be present in 15% or 30% proportions have been considered to indicate the presence of two nonallelic Hb_α structural loci.     

A rapid, hygienic method for the preparation of fecal samples for liquid scintillation counting.

A method for the preparation of fecal samples for liquid scintillation counting is described which is rapid, hygienic, and inexpensive. By the use of a novel type of homogenizer, fecal samples can be homogenized while totally enclosed within a sealed, plastic bag, so reducing the possible risk of infection. The subsequent preparation of a clear solution suitable for liquid seintillation counting is performed using an “in-vial” digestion technique which enables any ¹⁴CO₂ released during digestion to be trapped within the vial.     

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.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.