Triple-label beta liquid scintillation counting.

The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.     

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.     

Sources of error in the channels ratio method for efficiency determination in liquid scintillation counting

The reliability of the channels ratio method for determining counting efficiency in liquid scintillation counting was investigated. It was found that the efficiency of counting gels, cloudy samples, two-phase samples, samples in which the radioactive material was precipitating, and samples on solid supports could not be reliably determined from a normal quench correction curve. A curve constructed from external standard channels ratios was unreliable when mixing different vial sizes and sample volumes, but one constructed from sample channels ratios was not. It was also found that variation in instrument performance can result in large errors unless samples and standards are counted together. Statistical error changed relatively little within the range of ratios 0.3 to 0.8.     

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.     

New Instruments for High Throughput Receptor Binding Assays

Abstract

The TopCount^(R) Microplate Scintillation Counter and the Matrix 9600^(R) Direct Beta Counter are microplate compatible instruments developed to meet the needs of investigators using radioisotope assays adapted for very high throughput. This paper describes these instruments and their application to receptor binding assays. When combined with the appropriate sample handling equipment and filter media, use of these multi-detector instruments improves sample handling efficiency and shortens overall counting time. The assay protocols including filtration through glass fiber mats and membrane filters have been investigated. Results obtained from these new instruments are compared to standard techniques using conventional liquid scintillation and gamma counting.     

Use of methyl green-DNA agarose for detecting deoxyribonuclease activity in polyacrylamide gels

A liquid scintillation counting system is described that allows recovery of compounds for further study and analysis. For most classes of compounds tested (with the exception of steroids) the recovery was high (usually at least 90%) and in the case of nucleosides was accompanied by very little degradation of the sample. The counting method should be useful for the counting of samples where a high recovery of the compound is necessary.     

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.     

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.     

Exclusion of the sample sorption effects on the instability of liquid scintillation by emulsion counting

In activity assays of labeled compounds by liquid scintillation spectrometer, the effects due to sample sorption to the counting vial may be excluded by the use of the Triton X-100-toluene-based mixture in a 1:2 ratio by volume. For the ratio (v/v) of water to this mixture within the interval 1:4 to 1:1, the counting rates in particular channels, corrected to the disintegration half-time, are constant.     

Solid Scintillators for Receptor Assays: An Environmentally Safe Alternative to Liquid Scintillation Cocktails

Abstract

Scintillation counting of tritiated ligands is widespread in receptor assays and has necessitated the use of scintillation cocktails containing environmentally damaging solvents that pose health hazards to their users. A safer mode of dry scintillation counting, based on the solid scintillator Xtalscint, was evaluated in whole-cell and membrane receptor assays. The results compared favorably with those obtained with glass-fiber filters and conventional liquid counting methods. It is concluded that solid scintillators may be used as an environmentally safer alternative to liquid scintillation in these assays.     

A biuret method for determination of protein in hyamine hydroxide and NCS solutions used for liquid scintillation counting in toluene

A procedure for measurement of protein concentration in Hyamine hydroxide or NCS solution is described. The assay is linear from 0.5 to 8.0 mg. Use of this procedure offers the advantage of allowing liquid scintillation counting in toluene and protein assay on aliquots of the same sample.     

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.     

Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: a general solution of the problem

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of ³H and ¹⁴C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.     

Combustion and nonaqueous titration determination of carbon in plant tissues

A method for determination of total carbon and carbon-14 in biological samples by combustion and subsequent absorption and titration of carbon dioxide in organic amine is described. Results obtained with this method are comparable to those obtained with a carbon analyzer. Fifteen carbon-content determinations and sample preparations for carbon-14 liquid scintillation counting can be performed in 1 hr using this procedure.     

A rapid micromethod for the determination of nitrogen and phosphate in biological material

Two types of small containers for the liquid scintillation counting of 0.15–2 ml of aqueous sample in emulsifying agents have been investigated. Counting efficiency and reproducibility were found to be comparable with those found with more orthodox methods, but the effective cost is less. The systems also provide an economical way of using expensive reagents.     

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.     

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 carbon dioxide collection accessory for the rapid combustion apparatus for preparation of biological samples for liquid scintillation analysis

A carbon dioxide collection accessory is described for the previously reported combustion system for preparation of biological samples for liquid scintillation counting. A sample can be burned and collected every 3 min. Sample size is limited by the capacity of the solvent to absorb carbon dioxide without precipitation. The system can be used for collecting water as well as carbon dioxide, for double isotope counting. The method has a collection recovery of 97%, is calibrated by internal standards, and shows a coefficient of variation of 1.1%.     

Analysis of radioactive and nonradioactive purine bases,nucleosides, and nucleotides by high-speed chromatography on a single column

The concentrations of radioactive and nonradioactive purine bases, purine nucleosides, purine mono-, di-, and trinucleotides in acid extracts of fibroblasts were determined by anion-exchange column chromatography. The concentrations of nonradioactive components were determined by computerized integration of the signal from a double-beam uv-detector. The radioactive metabolites were quantitated by high-efficiency, continuous liquid scintillation counting, employing a discrete sample transport system.     

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.