The GTPγS binding assay was used to measure G-protein activation, as previously described (33 (link)). Briefly, 25 µg of CHO-hCB1-Rx or 50 µg of CHO-hCB1-Rx and CHO-hMOR membrane homogenates were added to reaction mixtures containing 10 mM MgCl2, 20 mM HEPES, 100 mM NaCl, 0.1 nM [35S]GTPγS, 10 µM GDP, 0.1% bovine serum albumin and the specified concentrations of ligand to be examined. The total volume of the incubation mixture was 1 ml. After mixing each solution, reaction mixtures were incubated at 30°C for 30 min. Nonspecific binding was defined by the amount of radioactivity remaining in the presence of 1 µM non-radiolabeled GTPγS. Reactions were terminated by rapid vacuum filtration through glass fiber filters followed by four washes with 5 ml of ice cold 50 mM HEPES (pH 7.4) containing 0.1% bovine serum albumin. Four ml of Scintiverse© scintillation fluid (Fisher Scientific, Waltham, MA) was added to the filters and the amount of radioactivity was quantified 24 hr later utilizing a Packard-Tri-carb 2100/TR liquid scintillation counter (PerkinElmer, Shelton, CT).
Scintiverse scintillation fluid
Manufactured by Thermo Fisher Scientific
Scintiverse© is a scintillation fluid manufactured by Thermo Fisher Scientific. Scintillation fluids are used in liquid scintillation counting, a technique that measures radioactivity by detecting the light emitted when radiation interacts with the fluid. Scintiverse© is formulated to provide efficient light production and high counting performance for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Glass fiber filters, by Brandel Inc (3 mentions)
Packard tri carb 2100 tr liquid scintillation counter, by PerkinElmer (2 mentions)
Tritiated thymidine, by PerkinElmer (1 mentions)
Dneasy miniprep kit, by Qiagen (1 mentions)
Taq polymerase, by New England Biolabs (1 mentions)
Beckman scintillation counter, by Beckman Coulter (1 mentions)
Hpaii, by New England Biolabs (1 mentions)
6 protocols using scintiverse scintillation fluid
1
GTPγS Binding Assay for G-Protein Activation
2
Splenocyte Mitogenesis Assay Protocol
3
Cytosine Methylation Analysis by Extension
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The methylation state of cytosines within genomic DNA was analyzed using a cytosine extension method [59 (link)]. Cellular genomic DNA was extracted from logarithmically growing CEM-C1-15, CEM-IV B9, RPMI 8226, RPMI-II E6, or Molt-4 cells treated with DMSO vehicle or 100 nM AZA for 48 hours using a Qiagen DNeasy mini-prep kit (Qiagen, Valencia, CA). Residual RNA contamination was eliminated by digestion with 400 μg of RNase A. Purified DNA was quantified and 2 μg was digested overnight at 37°C using 8 units of the methylation-sensitive restriction enzyme Hpa II (New England Biolabs, Ipswich, MA). Extension was performed by combining 0.25 μg of digested DNA in the presence of 3H deoxycytidine triphosphate (dCTP) (53.0 Ci/mmol, Perkin Elmer, Waltham, MA), 0.25 units of TAQ polymerase (New England Biolabs), and buffer supplied with the enzyme for 1 hour at 56°C. Samples were subsequently placed on ice and duplicate 10 μl aliquots were bound onto Whatman DE-81 ion exchange filter papers (Whatman Inc., Florham Park, NJ) using a vacuum manifold. Filters were washed 3 times with 0.5 ml room temperature PBS, air dried, submerged in 3 mls of Scintiverse scintillation fluid (Fisher Scientific, Fair Lawn, NJ), vigorously vortex mixed for 15 seconds, allowed to recover for 1 hour, and evaluated using a Beckman scintillation counter (Beckman Coulter).
4
CB1 and CB2 Receptor Binding Assay
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Competition receptor binding was performed as reported earlier [30 (link)]. Briefly, each reaction mixture contained either 100 μg of CHO-hCB1-Rx or 50 μg of CHO-hCB2 membrane homogenates, 0.2 nM [3H]-CP55,940, 5 mM MgCl2, and increasing concentrations of the non-radioactive competing ligands in a 50 mM Tris-HCl buffer (pH 7.4) with 0.1% bovine serum albumin. The total volume of the incubation mixture was 1 ml. All reactions were mixed and allowed to reach equilibrium binding by incubation at room temperature for 90 min. Non-specific binding was defined as the amount of radioligand binding remaining in the presence of a 1 μM concentration of the non-radioactive, high affinity, CB1/CB2 agonist WIN-55,212–2. Binding was terminated by rapid vacuum filtration through glass fiber filters (Brandel, Gaithersburg, MD), followed by four 5 ml washes of ice-cold 50 mM Tris-HCl (pH 7.4) buffer containing 0.1% bovine serum albumin. Four ml of scintiverse scintillation fluid (Fisher Scientific, Waltham, MA) was added to the filters and the amount of radioactivity was quantified 24 hr later utilizing liquid scintillation spectrophotometry.
5
GTPγS Binding Assay for G-Protein Activation
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The GTPγS binding assay to measure G-protein activation was performed as previously described [30 (link)]. Briefly, in a total volume of 1 ml, 25 μg of CHO-hCB2, 50 μg of CHO-hCB1-Rx or 50 μg of CHO-hMOR membranes homogenates were added to each reaction mixture containing 0.1 nM [35S]GTPγS, 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 10 μM GDP, 0.1% bovine serum albumin and the indicated concentrations of ligand to be examined. After mixing, reaction mixtures were incubated at 30°C for 30 min. (a time interval shown to produce optimal agonist-induced [35S]GTPγS binding levels, data not shown). Nonspecific binding was defined by the amount of radioactivity remaining in the presence of 10 μM non-radiolabeled GTPγS. Reactions were terminated by rapid vacuum filtration through glass fiber filters followed by four washes with ice cold 50 mM HEPES (pH 7.4) containing 0.1% bovine serum albumin. Four ml of scintiverse scintillation fluid (Fisher Scientific, Waltham, MA) was added to the filters and the amount of radioactivity was quantified 24 hr later utilizing liquid scintillation spectrophotometry.
6
Competitive Cannabinoid Receptor Binding
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