The effect of CaM (calmodulin) on [3H]ryanodine binding was assessed as described.37 (link),38 (link) SR-enriched vesicles were obtained from IZ and NIZ, using Tris-buffered solution (0.9% NaCl, 10 mmol/L Tris-HCl, pH 6.8) plus protease inhibitors (PIs) and collected by 3 steps of centrifugation. The 40 000-g pellets were resuspended in Tris-buffered solution with PI, plus 10% sucrose. After protein quantification, the [3H]ryanodine-bind-ing assay was performed. Briefly, 100 μl of [3H]ryanodine-binding buffer containing 200 mmol/L KCl, 25 mmol/L Tris, 50 mmol/L Hepes (pH 7.4), 1 mmol/L EGTA, 5 nmol/L [3H]ryano-dine (68.4 Ci-mmol-1; Dupont NEN), and CaCl2 was added to set free [Ca2+] to pCa 5. Ca2+/EGTA ratio was calculated with Max-Chelator (www.stanford.edu/~cpatton/maxc.html ), and 50 μg of SR-enriched solution was incubated for 2 hours at 37°C, filtered on GF/B glass filters (Whatman) presoaked with water, and washed twice with 5 mL of distilled water with an M24-R cell harvester (Brandel). Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine and subtracted from each sample.
M24r cell harvester
Manufactured by Brandel Inc
Sourced in United States
The M24R cell harvester is a laboratory instrument designed for the automated separation and collection of cells from liquid samples. It utilizes a centrifugation process to isolate the cells of interest, allowing for efficient and consistent cell harvesting.
Automatically generated - may contain errors
Lab products found in correlation
Ls6500, by Beckman Coulter (7 mentions)
Whatman gf c glass fiber filters, by Cytiva (5 mentions)
Gf b glass filter, by Brandel Inc (3 mentions)
3h ryanodine, by PerkinElmer (2 mentions)
Tricarb 2300tr, by Hewlett-Packard (2 mentions)
Gf b glass fiber, by Cytiva (2 mentions)
Gf b glass filters, by Cytiva (1 mentions)
Gf c glass fiber filter, by Cytiva (1 mentions)
Prism 5, by GraphPad (1 mentions)
Whatman gf c glass filters, by Cytiva (1 mentions)
3h pib, by American Radiolabeled Chemicals (1 mentions)
Skf81297, by Bio-Techne (1 mentions)
Gf b filters, by Brandel Inc (1 mentions)
Glass fiber gf c filters, by Cytiva (1 mentions)
Gf c glass filters, by Cytiva (1 mentions)
Gf b glass fiber filters, by Cytiva (1 mentions)
Whatman gf b glass fiber filters, by Cytiva (1 mentions)
21 protocols using m24r cell harvester
1
CaM Regulation of Ryanodine Receptor Binding
2
In Vitro Opioid Receptor Binding Assays
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In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures [51 (link)]. Assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL. Cell membranes (20 µg) were incubated with various concentrations of test compounds of [3H]U69,593 (0.4 nM) or [3H]diprenorphine (0.2 nM) for labeling KOR or DOR, respectively, for 60 min at 25 °C. Non-specific binding was determined using 10 µM U69,593 or 1 µM diprenorphine. After incubation, reactions were terminated by rapid filtration through Whatman GF/C glass fiber filters. Filters were washed three times with 5 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester (Brandel, Gaithersburg, MD, USA). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA, USA). Inhibition constant (Ki, nM) values were determined by the method of Cheng and Prusoff [66 (link)] from concentration–response curves by nonlinear regression analysis using the GraphPad Prism 5.0 Software (GraphPad Prism Software Inc., San Diego, CA, USA). All experiments were performed in duplicate and repeated at least three times with independently prepared samples.
3
Ryanodine Receptor Binding Assay
4
Quantifying Brain Amyloid Binding
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Unfractionated whole brain tissue homogenates (described above) were diluted from 300 mg/ml to a concentration 10 mg/ml in PBS prior to the binding assay as previously described (Ikonomovic et al., 2008 (link)) with the exception of the fold-higher initial homogenate prepared in the current study. For determination of 3H-PiB binding, 1 nM 3H-PiB (American Radiolabeled Chemicals, St. Louis, MO, USA; specific activity 72.4 Ci/mmol) was incubated with 100 μg tissue in 1 ml PBS as described previously (Ikonomovic et al., 2008 (link)). Unlabeled PiB was dissolved in DMSO at 400 mM (to yield 51% DMSO) and this stock solution was diluted with PBS to achieve the desired concentration for the binding assay. Non-specific binding was defined as the number of counts remaining in the presence of 1 mM unlabeled PiB. The binding mixtures were filtered through a Whatman GF/B glass filter using a Brandel M24R cell harvester (Brandel, Gaithersburg, MD) and rapidly washed five times with 3 ml PBS. The filters were counted in Cytoscint-ES after thorough vortex mixing and resting overnight. Results were corrected for non-specific, non-displaceable binding in the presence of 1 mM PiB and expressed as picomoles 3H-PiB bound per gram of wet brain tissue weight in the homogenate.
5
Optimized D1 Receptor-Stimulated GTPγS Binding
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The functional [35S]GTPγS binding experiments were performed as previously described59 (link),60 (link), with modifications for optimizing the binding assay stimulated with a D1R agonist61 (link). Briefly the membrane homogenates were incubated at 30 °C for 60 min in buffer (pH 7.4) composed of 25 mM HEPES, 120 mM NaCl, 20 mM MgCl2, 1.8 mM KCl, and 1 mM sodium deoxycholate containing 20 MBq/0.05 cm3 (link) [35S]GTPγS (0.05 nM) and increasing concentrations (10—10 (link) to 10—5 (link)M) of the selective D1R full agonist, SKF81297 (Tocris Bioscience, Bristol, United Kingdom)62 (link). The experiments were performed in the presence of excess GDP (10 µM) in a final volume of 1 mL. Total binding was measured in the absence of test compounds, whereas non-specific binding was determined in the presence of 10 µM unlabeled GTPγS and subtracted from the total binding. The difference represented basal activity. The reaction was terminated by rapid filtration under vacuum (Brandel M24R Cell Harvester), and washed three times with 5 mL of ice-cold 0.1 M phosphate (pH 7.4) buffer through Whatman GF/B glass fibers. The radioactivity of the dried filters was detected in an UltimaGold MV aqueous scintillation cocktail on a Packard Tricarb 2300TR liquid scintillation counter. [35S]GTPγS binding experiments were performed in triplicate and were repeated at least three times.
6
Dissociation Kinetics of 18F-5-OH-FPPAT
7
In Vitro Binding Assay for AV-1451
8
Opioid Receptor Binding Assays
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Binding
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
9
Competitive Binding Assay for Brain Receptors
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In competitive binding experiments, we used radio-labelled ligands in increasing concentrations and measured the amount of specifically bound radioactive ligands in the function of the applied radio-ligand concentrations.
Aliquots of frozen rat and guinea pig brain membrane homogenates were centrifuged (20 min, 18,000 rpm, 4 °C) to remove sucrose, and the pellets were suspended in 50 mM Tris-HCl buffer (pH 7.4). Membranes were incubated in the presence of the unlabelled DAMGO, HS665, and CART peptides in increasing concentrations (10−10–10−5 M) at 35 °C for 45 min with [3H]DAMGO and 30 °C for 45 min with [3H]HS665. The non-specific and total binding were determined in the presence and absence of 10 µM unlabelled naloxone and HS665, respectively. The reaction was terminated by rapid filtration under vacuum (Brandel M24R Cell Harvester) and washed three times with 5 mL ice-cold 50 mM Tris-HCl (pH 7.4) buffer through Whatman GF/C glass fibres. The radioactivity of the dried filters was detected in an UltimaGoldTM MV aqueous scintillation cocktail with Packard Tricarb 2300TR liquid scintillation counter. The competitive binding assays were performed in duplicate and repeated at least three times.
Aliquots of frozen rat and guinea pig brain membrane homogenates were centrifuged (20 min, 18,000 rpm, 4 °C) to remove sucrose, and the pellets were suspended in 50 mM Tris-HCl buffer (pH 7.4). Membranes were incubated in the presence of the unlabelled DAMGO, HS665, and CART peptides in increasing concentrations (10−10–10−5 M) at 35 °C for 45 min with [3H]DAMGO and 30 °C for 45 min with [3H]HS665. The non-specific and total binding were determined in the presence and absence of 10 µM unlabelled naloxone and HS665, respectively. The reaction was terminated by rapid filtration under vacuum (Brandel M24R Cell Harvester) and washed three times with 5 mL ice-cold 50 mM Tris-HCl (pH 7.4) buffer through Whatman GF/C glass fibres. The radioactivity of the dried filters was detected in an UltimaGoldTM MV aqueous scintillation cocktail with Packard Tricarb 2300TR liquid scintillation counter. The competitive binding assays were performed in duplicate and repeated at least three times.
10
Ryanodine Binding Assay for Skeletal SR
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[3H]Ryanodine binding to rabbit skeletal SR was performed as described previously (El-Hayek et al., 1995 (link); Gurrola et al., 1999 (link); Schwartz et al., 2009 (link)). In brief, calcins were diluted directly into the incubation medium (0.2 M KCl, 10 µM CaCl2, and 10 mM Na-HEPES, pH 7.2) to achieve a final concentration of 1 pM to 20 µM, as indicated in graphs. To determine the effect of calcins on Ca2+ sensitivity of [3H]ryanodine binding activity, each calcin was added at fixed concentration (100 nM) to the standard incubation medium, which contained 0.2 M KCl, 1 mM Na2EGTA, 10 mM Na-PIPES, pH 7.2, and CaCl2 to set free [Ca2+] in the range of 10 nM to 10 mM. The Ca2+/EGTA ratio was calculated with the computer program MAXCHELATOR. [3H]Ryanodine (95 Ci·mmol−1; PerkinElmer) was diluted directly in the incubation medium to a final concentration of 5 nM. Protein concentration of heavy SR was 0.3 mg·ml−1 and was determined by the Bradford method. Incubation lasted 120 min at 36°C. 100-µl samples were always run in duplicate, filtered on GF/C glass filters (Whatman) and washed twice with 5 ml of distilled water using an M24-R cell harvester (Brandel). Nonspecific binding was determined in the presence of 20 µM unlabeled ryanodine, reached no more than 5–10% of the total binding, and was subtracted from each sample.
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