[3H]-thymidine (Perkin Elmer NET355001MC, PerkinElmer, Waltham, MA) was utilized for in vitro assessment of therapy-induced changes in thymidine salvage pathway activity in cultured human NSCLC cells. [3H]-thymidine specific activity was >10Ci(370GBq)/mmol and radiochemical purity >97%. H460 and H1299 cells were seeded (1×106/well) in 6-well plate in RPMI1640 supplemented with 10% FBS and antibiotics, incubated 24 hours in 5% CO2 at 37°C. When cell cultures achieved 80% confluence, cells were exposed to treatment with either the vehicle (sterile water), pemetrexed (100 nM), or the combination of pemetrexed (100 nM) and cisplatin (10 μM) in growth media for varying exposure times ranging up to 48 hours. Drug-containing medium was then removed, and the cells were then washed and pulsed with 5 μCi [3H]-thymidine/well for 1 hour. The cells were then washed and scraped into plastic vials. Scintillant (10 mL; Research Products International Corp., Mount Prospect, IL) was added to each vial and the radioactivity was counted on a scintillation counter (Beckman Coulter LS6500, Beckman Coulter Life Sciences, Indianapolis, IN).
Ls6500
Manufactured by Beckman Coulter
Sourced in United States, Germany, United Kingdom, Canada, China
The LS6500 is a fully automated, multi-purpose flow cytometer designed for a wide range of applications in clinical diagnostics and research laboratories. It features advanced technology for precise cell analysis and sorting, delivering accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
M24r cell harvester, by Brandel Inc (7 mentions)
Ultima gold, by PerkinElmer (7 mentions)
Whatman gf c glass fiber filters, by Cytiva (5 mentions)
Scintisafe 30, by Thermo Fisher Scientific (4 mentions)
Sampling manifold, by Merck Group (4 mentions)
L 3h glutamate, by PerkinElmer (4 mentions)
Ultima gold xr, by PerkinElmer (4 mentions)
Scintillation cocktail, by Thermo Fisher Scientific (3 mentions)
Glucose transporter inhibitor 2, by Merck Group (3 mentions)
3h thymidine, by Beckman Coulter (3 mentions)
3h cholesterol, by PerkinElmer (3 mentions)
35s methionine, by PerkinElmer (3 mentions)
Net043001mc, by PerkinElmer (3 mentions)
Emulsifier safe, by PerkinElmer (3 mentions)
3h thymidine, by PerkinElmer (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Surveyor hplc system, by Thermo Fisher Scientific (3 mentions)
Net355001mc, by PerkinElmer (2 mentions)
Tissue solubilizer, by Cytiva (2 mentions)
Scintiverse, by Thermo Fisher Scientific (2 mentions)
328 protocols using ls6500
1
Thymidine Salvage Pathway Assay in NSCLC
2
Binding Assays of Opioid Ligands
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Binding assays were conducted on human MOR stably transfected into CHO cells (CHO-hMOR) according to the published procedure [43 (link)]. Cell membranes were prepared as described previously, and stored at −80 °C until use [43 (link)]. Protein concentration of cell membrane preparations was determined by the method of Bradford using bovine serum albumin as the standard [49 (link)]. Cell membranes (15–20 µg) were incubated in 50 mM Tris-HCl buffer (pH 7.4) with [3H]DAMGO (1 nM) and various concentrations of test peptides in a final volume of 1 mL, for 60 min at 25 °C. Non-specific binding was determined using 10 µM of unlabeled DAMGO. After incubation, reactions were terminated by rapid filtration through Whatman glass GF/C 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 (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). The inhibitory constant (Ki, in nM) values were calculated from the competition binding curves by nonlinear regression analysis and the Cheng–Prusoff equation [50 (link)]. All experiments were performed in duplicate and repeated at least three times.
3
Comparing Liquid Scintillation Counting Systems
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The sources were serially counted in all three of the commercial LS counting systems maintained by our group: a Beckman Coulter LS6500 (Beckman Coulter, Pasadena, CA), a Packard 2500TR, and a Wallac 1414 Guardian (both Perkin Elmer, Waltham, MA). Each source set was counted for 10 cycles of between 600 s and 900 s before being moved to the next counter. The total number of counts in each spectrum (open window) was more than 106. The sources were agitated before being inserted into each counter to ensure proper mixing.
Although the CNET technique was applied to these samples, no 3H tracing sources were prepared. Instead, efficiencies were calculated based on model calculations with estimated 3H efficiencies (see Sec. 3.4.1).
Although the CNET technique was applied to these samples, no 3H tracing sources were prepared. Instead, efficiencies were calculated based on model calculations with estimated 3H efficiencies (see Sec. 3.4.1).
4
Opioid Receptor Binding Assay Protocol
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Competitive binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures [26 (link),27 (link)]. Binding assays were performed using [3H]DAMGO (1 nM), [3H]diprenorphine (0.2 nM), [3H]U69,593 (0.4 nM), or [3H]Nociceptin (0.1 nM) for labeling MOR, DOR, KOR, or NOP receptors, respectively. Non-specific binding was determined using 1–10 µM of the unlabeled counterpart of each radioligand. Assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL. In NOP receptor experiments, 1 mg/mL bovine serum albumin (BSA) was added to the assay buffer. Cell membranes (15–20 µg) were incubated with various concentrations of test compounds and the appropriate radioligand for 60 min at 25 °C. After incubation, reactions were terminated by rapid filtration through Whatman GF/C glass fiber filters. In the NOP receptor assay, filtration was carried out through 0.5% PEI-soaked 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 (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). All experiments were performed in duplicate and repeated three times with independently prepared samples.
5
Thymidine Salvage Pathway Assay in NSCLC Cells
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[3H]-thymidine (Perkin Elmer NET355001MC, PerkinElmer, Waltham, MA) was utilized for in vitro assessment of therapy-induced changes in thymidine salvage pathway activity in cultured human NSCLC cells. [3H]-thymidine specific activity was >10Ci(370GBq)/mmol and radiochemical purity >97%. H460 and H1299 cells were seeded (1×106/well) in 6-well plate in RPMI1640 supplemented with 10% FBS and antibiotics, incubated 24 hours in 5% CO2 at 37°C. When cell cultures achieved 80% confluence, cells were exposed to treatment with either the vehicle (sterile water), pemetrexed (100 nM), or the combination of pemetrexed (100 nM) and cisplatin (10 μM) in growth media for varying exposure times ranging up to 48 hours. Drug-containing medium was then removed, and the cells were then washed and pulsed with 5 μCi [3H]-thymidine/well for 1 h. The cells were then washed and scraped into plastic vials. Scintillant (10 ml; Research Products International Corp., Mount Prospect, IL) was added to each vial and the radioactivity was counted on a scintillation counter (Beckman Coulter LS6500, Beckman Coulter Life Sciences, Indianapolis, IN). For ENT1 inhibition, cells were incubated with NBMPR (Nitrobenzylthioinosine) for 15min (100uM; Sigma-Aldrich, St. Louis, MO) prior to labeling with [3H]-thymidine.
6
Biodistribution of Radiolabeled Sunitinib
7
Biodistribution of Radiolabeled PLGA NPs
8
Radiolabeled Tracer Efficiency Calculations
9
Quantifying Tracer Extraction from Wood
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After the exposure, wood samples were sheared with a hammer and knife into four equal parts (~1.25 × 2.5 × 1.5 cm 3 ), where parts A and D were those originally located next to the end-grain side whereas parts B and C were the two middle sections (Figure 1). Each wood part was further sheared into smaller pieces to allow for an exhaustive TAZ extraction, which was carried out for 16 hours using a Soxhlet apparatus with 130.0 mL of acetone. The extract was evaporated to dryness using a vacuum rotary evaporator and reconstituted in 1.5 mL of acetone. For the liquid scintillation counting analysis, 1.5 mL of an acetone dissolved sample was vortexed with 3.5 mL of the scintillation cocktail and analyzed after an overnight equilibration in a scintillation counter, Beckman Coulter LS 6500, purchased from Beckman Coulter, Inc. (Fullerton, CA, USA). The analysis was run in duplicate for 10.0 min in a DPM calculation mode (disintegrations per minute, which is directly proportional to the tracer concentration).
Water run-off was collected several times per week and the volume was recorded.
As with the wood extracts, run-off water was evaporated to dryness using a vacuum rotary evaporator and reconstituted in 1.5 mL of acetone, which was analyzed using a scintillation counting analysis as described previously.
Water run-off was collected several times per week and the volume was recorded.
As with the wood extracts, run-off water was evaporated to dryness using a vacuum rotary evaporator and reconstituted in 1.5 mL of acetone, which was analyzed using a scintillation counting analysis as described previously.
10
Quantifying Cellular Lipid and Histone Labeling
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BN cells were cultured with 0.5 µCi of [ 3 H]-acetyl-CoA (Perkin Elmer, Waltham, MA) in 1 mL of complete media for 24 hours. Media was collected in separate tubes and the pellets were washed three times with PBS. The cell pellets were fully solubilized with 200 µl of a 3% solution of potassium hydroxide (KOH) (29) . Solubilized cell pellets were then extracted for their lipid using 1ml of a 2:1 (v/v) chloroform:methanol solution, similar to the Folch method (without the neutralization step with acid/chloride salt) (Folch, Lees, & Stanley, 1957) . Histones were prepared directly from cell pellets using a histone extraction kit according to the manufacturer's instructions (Abcam, Toronto, ON). Disintegrations per minute (DPM) were determined using a LS6500 scintillation counter (Beckman Coulter, Ramsey, MN) using 10 µl from either solubilized cell pellets, lipid extracts and histone extracts.
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