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Ls6500 scintillation counter

Manufactured by Beckman Coulter
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The LS6500 scintillation counter is a laboratory instrument used for the detection and measurement of radioactive samples. It is designed to quantify the amount of radioactive material present in a sample by measuring the light pulses generated when the sample interacts with a scintillation material. The LS6500 provides accurate and reliable results for a wide range of applications in fields such as biochemistry, environmental analysis, and nuclear research.

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Lab products found in correlation

T4 polynucleotide kinase pnk, by PerkinElmer (3 mentions) γ 32p atp 5 triphosphate, by PerkinElmer (3 mentions) 3h thymidine, by PerkinElmer (3 mentions) Ecolite liquid scintillation cocktail, by MP Biomedicals (3 mentions) Plate rotator, by Harvard Apparatus (3 mentions) Scintillation fluid, by Thermo Fisher Scientific (3 mentions) 3h 2 deoxy d glucose, by PerkinElmer (2 mentions) Cytochalasin b, by Merck Group (2 mentions) 2 deoxy d glucose, by PerkinElmer (2 mentions) 3h sam, by PerkinElmer (2 mentions) Anti ha agarose beads, by Merck Group (2 mentions) C18 reverse phase column, by Macherey-Nagel (2 mentions) Frc 10a fraction collector, by Beckman Coulter (2 mentions) Nec084h001mc, by PerkinElmer (2 mentions) L 4 5 3h n leucine, by PerkinElmer (2 mentions) Radiolabeled nabh4, by Moravek Biochemicals (2 mentions) 5 kda mwco 6 well equilibrium dialyzer, by Harvard Apparatus (2 mentions) Q exactive hybrid quadrupole orbitrap, by Thermo Fisher Scientific (2 mentions) Complete counting cocktail, by Research Products International (2 mentions) 2 dog, by Merck Group (2 mentions)

134 protocols using ls6500 scintillation counter

1

Measuring Purine and Pyrimidine Metabolism

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Actively proliferating CUTLL1 or Jurkat cells were switched to 10% dialyzed FBS-containing medium and incubated for 1 hour. Cells were then labeled with 2 μCi of the specific activity of either 14C-glycine (for de novo purine pathway), 3H-hypoxanthine (for salvage purine pathway), 14C-aspartate (for de novo pyrimidine pathway), or 3H-uridine (for pyrimidine salvage pathway) for 6 hours. RNA was isolated using an RNA extraction kit (Qiagen, 74106). For each sample, out of 100 μl of eluted RNA, 70 μl was mixed with 3 ml of scintillation fluid and counts per minute (CPM) was taken in a Beckman LS6500 scintillation counter. CPM was normalized with the total RNA from each sample.
To measure the uptake of glycine, hypoxanthine, aspartate, and uridine, the cells were labeled with 1 μCi of the specific activity of 14C-glycine, 3H-hypoxanthine, 14C-aspartate, or 3H-uridine for 5 min. Plates were then kept on ice to stop the uptake followed by two washes with chilled phosphate-buffered saline (PBS) and lysed in 0.5 ml of protein lysis buffer. 400 μl lysate was mixed with 3 ml of scintillation fluid, and the count was taken in a Beckman LS6500 scintillation counter. CPM was normalized with respective protein concentrations. All the conditions were analyzed with at least three independent biological replicates.
Srivastava S., Sahu U., Zhou Y., Hogan A.K., Sathyan K.M., Bodner J., Huang J., Wong K.A., Khalatyan N., Savas J.N., Ntziachristos P., Ben-Sahra I, & Foltz D.R. (2021). NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia. Science Advances, 7(5), eabc9781.
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2

Cell Proliferation Assay with [3H] Thymidine

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Radioactively labelled (tritium) thymidine was used to measure the proliferation of MDA-MB-231 cells by the incorporation of [3H] thymidine into the DNA of dividing cells. Cells were seeded at a seeding density of 2 × 104 cells in 24 well plates. Following treatment, cells were labelled with 0.1 μCi [3H] thymidine per well, incubated with 5% trichloroacetic acid at 4°C for 10 minutes, followed by a 1 hour incubation with 1 M sodium hydroxide. The resulting suspension was added to a vial containing 2 mL ultima gold liquid scintillation cocktail (Perkin Elmer Beaconsfield, Bucks, UK) and incorporated counts were measured using a Beckman Scintillation Counter LS6500. Data were recorded as disintegrations per minute (DPM).
Shafei M.A., Forshaw T., Davis J., Flemban A., Qualtrough D., Dean S., Perks C., Dong M., Newman R, & Conway M.E. (2020). BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer. Oncotarget, 11(21), 1971-1987.
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3

Quantifying Intracellular Radioactive Substrates

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Intracellular concentrations of radioactively labeled substrates MPP+ and TEA+ were quantified using liquid scintillation counting. To this end, 400 μl cell lysate was transferred into a 20 ml scintillation vial, 9 ml Aquasafe 500 Plus liquid scintillator (both Zinsser Analytics) was added, and the sample was measured in the Scintillation Counter LS6500 (Beckman Coulter).
Meyer M.J., Schreier P.C., Basaran M., Vlasova S., Seitz T., Brockmöller J., Zdrazil B, & Tzvetkov M.V. (2022). Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1. The Journal of Biological Chemistry, 298(6), 101974.
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4

Measuring Sodium-Dependent Glutamate Uptake in Astrocytes

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Sodium-dependent transport activity was measured from lentivirus-transduced astrocytes on 12 well plates as described earlier (Li et al. 2006 (link), Ghosh et al. 2011 (link)). Cells were first rinsed with Na+ or choline-containing buffer (2×1 mL). Accumulation of radioactive L-[3H]-glutamate (0.5 μM) was measured in presence or absence of 300 μM dihydrokainate (DHK; Tocris Bioscience), a selective inhibitor of GLT-1 mediated glutamate uptake (Robinson et al. 1991 (link), Arriza et al. 1994 (link)). We previously showed that uptake is linear for at least 10 min under these conditions, and uptake was stopped after 5 min by rinsing the cells with ice-cold choline-containing buffer. After solubilization with NaOH, an aliquot of the cell extract was used for quantification of protein (Lowry et al. 1951 (link)) and a separate aliquot was used for analysis of radioactivity (Beckman LS 6500 scintillation counter). Dihydrokainate-sensitive Na+-dependent uptake was compared in control and transduced astrocytes (Ghosh et al. 2011 (link)).
Ghosh M., Lane M., Krizman E., Sattler R., Rothstein J.D, & Robinson M.B. (2015). Transcription Factor Pax6 Contributes to Induction of GLT-1 Expression in Astrocytes Through an Interaction with a Distal Enhancer Element. Journal of neurochemistry, 136(2), 262-275.
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5

GLUT-Specific Glucose Uptake Inhibition

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The inhibitory activity of DRB18 on glucose transport in cancer cells was analyzed by measuring the cell uptake of 2-deoxy-d-[3H] glucose as previously described [26 (link), 34 (link)].
For four GLUT-specific cell lines, HEK293 cells expressing one specific GLUT only per cell line [35 (link)] were a gift from Dr. Paul Hruz at Washington University of St. Louis. Cell plates were pre-treated with 25 μg/ml Polyethyleneimine for 20 min. The plates were then allowed to dry for 5–10 min after aspiration of PEI solution. A total of 50,000 cells were seeded into each well of the 24-well plate. After overnight incubation, cells were washed with KRP buffer twice and 225 μl of glucose-free KRP buffer was added. Additionally, mock or inhibitor was added to cells and incubated for 30 min. A mixture composed of 12.5 μl of 37 MBq/l 2-deoxy-D-[3H] glucose and 25 μl of 1 mmol/l regular glucose was added to cells to initiate glucose uptake. After 4 min, the cells were washed and lysed, and then radioactivity of the cell lysates was counted using a LS 6500 Scintillation Counter (Beckman Coulter). Glucose uptake assays in cancer cell lines were performed as previously described [25 , 34 (link)].
Shriwas P., Roberts D., Li Y., Wang L., Qian Y., Bergmeier S., Hines J., Adhicary S., Nielsen C, & Chen X. (2021). A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism. Cancer & Metabolism, 9, 14.
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6

Radiolabeling of Oligonucleotides using PNK

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T4 polynucleotide kinase (PNK) and γ-32P-ATP-5΄-triphosphate were obtained from Perkin Elmer. ONs were labeled by mixing PNK, PNK buffer, ATP, RNA and water (final volume = 50 μl) according to manufacturer's procedure followed by heating to 37°C for 45 min. Radiolabeled materials were passed through a G-25 sephadex column followed by purification via electrophoresis (20% denaturing PAGE). The bands of interest (slowest) were cut and eluted over a phosphate saline buffer solution (10 mM NaCl, 10 mM Na2P2O7, pH 7.2) for 6 h at 37°C. The remaining solution was filtered and concentrated to dryness under reduced pressure followed by precipitation over NaOAc and ethanol. Supernatant was removed and the remaining ON was dried and re-dissolved in water. Activity was assessed using a Beckmann LS 6500 scintillation counter.
Choi Y.J., Gibala K.S., Ayele T., Deventer K.V, & Resendiz M.J. (2016). Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA—a study of duplex, hairpins and the aptamer for preQ1 as models. Nucleic Acids Research, 45(4), 2099-2111.
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7

Quantifying Cellular Lipid Uptake

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Cells grown in 6-well plates were serum starved for 16h, with 2uCi/ml 14C-glucose added to the media for the final 3h. Cells were washed twice with phosphate buffered saline before lysis in 0.5% Triton X-100. Lipids were extracted with 3:2 (v/v) hexane/isopropanol (1 ml) and quantified in duplicate samples using a LS6500 scintillation counter (Beckman Coulter, Danvers, MA, USA), and normalized to protein concentration.
Li J., Yan H., Zhao L., Jia W., Yang H., Liu L., Zhou X., Miao P., Sun X., Song S., Zhao X., Liu J, & Huang G. (2016). Inhibition of SREBP increases gefitinib sensitivity in non-small cell lung cancer cells. Oncotarget, 7(32), 52392-52403.
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8

Radiolabeled Glucose Uptake Assay in Adipocytes

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Glucose uptake was measured as the incorporation of radiolabeled 2-deoxyglucosein day 7 3T3-L1 differentiated adipocytes. Briefly, 2 × 105 cells per well were grown in 12-well plates and serum-starved for 16 h. The cells were then washed twice with Krebs-Ringers Henseleit (KRH) buffer (20 mM HEPES pH 7.4, 136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO4, and 1.25 mM CaCl2) containing 0.1% bovine serum albumin (BSA) and incubated for 15 min in 0.45 ml KRH/BSA, followed by addition of 0.1 μM cold 2-deoxy-D-glucose and 0.5 μCi 2-deoxy-D-[3H]-glucose (Perkin Elmer) for 4 min. Following the incubation, cells were placed on ice, washed twice with ice-cold PBS, and lysed in 0.5 ml of 0.05% sodium dodecyl sulfate (SDS). The lysate (0.4 ml) was counted in 5 ml of scintillation fluid using a Beckman LS6500 scintillation counter. Rates of non-specific glucose uptake were determined in samples pre-treated for 10 min with cytochalasin B (10 μM, Sigma) and were subtracted from the total uptake. The specific glucose uptake was normalized to protein content.
Wagner G., Fenzl A., Lindroos-Christensen J., Einwallner E., Husa J., Witzeneder N., Rauscher S., Gröger M., Derdak S., Mohr T., Sutterlüty H., Klinglmüller F., Wolkerstorfer S., Fondi M., Hoermann G., Cao L., Wagner O., Kiefer F.W., Esterbauer H, & Bilban M. (2021). LMO3 reprograms visceral adipocyte metabolism during obesity. Journal of Molecular Medicine (Berlin, Germany), 99(8), 1151-1171.
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9

2-DG Uptake Assay in T. cruzi-Infected Fibroblasts

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NHDF were plated at 1 x 105 per well in 12 well plates and incubated with T. cruzi trypomastigotes at the indicated MOI or MOI of 40 for 1 hour. At 48 hpi monolayers were washed twice with Krebs-Ringer bicarbonate HEPES buffer (KRBH; 120 mM sodium chloride, 4 mM potassium phosphate monobasic, 1 mM magnesium sulfate, 0.75 mM calcium chloride, 30 mM HEPES, and 10 mM sodium bicarbonate) and incubated in 400 μL fresh KRBH at 37°C for 10 minutes. Transport assays were started by the addition of 0.5 μCi [3H]-2-DG and a final concentration of 55.5 μM unlabeled 2-DG and incubated at 37°C for 20 minutes. The reaction was stopped by addition of 35 μM cytochalasin B (MP Biomedicals) and monolayers were washed 5 times to remove residual 2-DG. All cells were lysed in 500 μL of 0.1 M sodium hydroxide at room temperature for 10 minutes, and 400 μL was added to 5 mL of Ecolite(+) (MP Biomedicals) for scintillation counting in a Beckman LS6500 scintillation counter. Background counts were removed by subtracting the measurements from samples where [3H]-2-DG and cytochalasin B were added simultaneously before immediate washing.
Shah-Simpson S., Lentini G., Dumoulin P.C, & Burleigh B.A. (2017). Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes. PLoS Pathogens, 13(11), e1006747.
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10

Protein Methyltransferase Assays in Vitro

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The procedures for cloning, expression and purification of proteins and protein complexes used for in vitro studies (enzymology, SEC-MALLS and X-ray crystallography) are described in supplementary materials. The tRNAs from S. cerevisiae or H. volcanii strains were purified as previously described (47 (link)).
The MTase assays were performed in a total volume of 10 μl (for HvoMtq2-Trm112) or 20 μl (for HvoTrm9–Trm112) containing 400 mM potassium phosphate buffer pH 7.5, 3 M KCl, 2.5 mM EDTA, 5 mM MgCl2, 5 mM NH4Cl, 0.25 mg/ml Bovine Serum Albumin, 50 μM S-adenosyl-l-methionine (SAM; containing 0.87 Ci/mmol of [3H]-SAM; Perkin Elmer) and 5 pmol of HvoMtq2–Trm112 complex or 2 pmol of HvoTrm9–Trm112 complex. The reaction was initiated by adding 100 pmol of substrate (HvoaRF1, HvoaRF3 or total tRNAs) to the mixture. After incubation for 2 h at 45°C, the reaction was stopped by precipitation with cold trichloroacetic acid (5%), followed by filtration on Whatman GF/C filters. The [3H] incorporation was measured using a Beckman Coulter LS6500 scintillation counter.
van Tran N., Muller L., Ross R.L., Lestini R., Létoquart J., Ulryck N., Limbach P.A., de Crécy-Lagard V., Cianférani S, & Graille M. (2018). Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes. Nucleic Acids Research, 46(16), 8483-8499.
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