Blood from six healthy volunteers (three males and three females) was collected as described above. Blood was mixed 1:1 with RPMI-1640 cell culture medium containing 1 µM 14C-UA and 1000 µM non-labelled UA. A total volume of 1790 µl of the mixture was pipetted into six well cell culture plates (BD Falcon) and incubated at 37 °C 5% CO2 on a shaker board. Incubation was terminated at 0, 0.5, 1, 2 and 4 h by transferring the cell suspensions to 2 ml microcentrifuge tubes (Eppendorf) and centrifuged at 2000 G for 10 min at 5 °C. Supernatants and cellular fractions were separated and hydrolyzed by incubation with tissue solubilizer “Solvable” at 55 °C for 3 hours according to manufacturers’ instructions. To the hydrolyzed cellular fractions, 50 µl of 0.2 M EDTA and 500 µl of 30% hydrogen peroxide were added and the mixtures were incubated at room temperature for 30 min, and then at 55 °C for 60 min in order to decolorize the samples. After cooling down, both fraction types were mixed with 17.7 ml “Ultima Gold” scintillation cocktail. Radioactivity (d.p.m.) was measured in a Packard Liquid Scintillation Counter. The extent of UA degradation was determined by counting radioactivity after addition to cell culture supernatants of ZnCl2 that precipitates only non-degraded UA.
Liquid scintillation counter
Manufactured by Hewlett-Packard
Sourced in United States, Switzerland
The Liquid Scintillation Counter is a laboratory instrument used to measure the radioactivity of liquid samples. It operates by detecting the light flashes, or scintillations, produced when ionizing radiation interacts with a scintillation solution containing the sample. The counter precisely measures the intensity and duration of these light flashes, providing quantitative data on the radioactive content of the sample.
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Lab products found in correlation
Six well cell culture plates, by BD (1 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Solvable, by PerkinElmer (1 mentions)
Irgasafe plus, by PerkinElmer (1 mentions)
Ultima gold, by PerkinElmer (1 mentions)
3h 5 ht, by PerkinElmer (1 mentions)
Ussing chamber, by Physiologic Instruments (1 mentions)
Rc dc protein assay kit, by Bio-Rad (1 mentions)
Tritiated thymidine, by PerkinElmer (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Spin x centrifuge tube filter, by Corning (1 mentions)
14 protocols using liquid scintillation counter
1
Measuring Uric Acid Degradation in Human Blood
2
Metabolic Labeling of Cell Lines and Primary Cells
3
Radiometric Analysis of Biological Samples
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Aliquots of plasma and urine samples were added directly to Irgasafe Plus (PerkinElmer) scintillation cocktail. Aliquots of blood, homogenized faeces and vomitus samples were pre‐treated with Solvable (PerkinElmer) tissue solubilizer before being added to the scintillation cocktail.
Radioactivity was determined using a Packard liquid scintillation counter equipped with disintegrations per minute (dpm) and luminescence options (eg Tri‐Carb 2500 TR, 2550 TR/LL or 2900 TR). All values were corrected with a background of 19 counts per minute (cpm). Based on a background value of 19 cpm, a counting time of 10 minutes and counting efficiencies of 85%‐95%, the limits of quantification were 20‐23 dpm. The limits of detection were 7‐8 dpm (one third of the limit of quantification). For blood and plasma samples, dpm/mL was based on measured dpm/g and assuming a density of approximately 1.0 g/mL.
Radioactivity was determined using a Packard liquid scintillation counter equipped with disintegrations per minute (dpm) and luminescence options (eg Tri‐Carb 2500 TR, 2550 TR/LL or 2900 TR). All values were corrected with a background of 19 counts per minute (cpm). Based on a background value of 19 cpm, a counting time of 10 minutes and counting efficiencies of 85%‐95%, the limits of quantification were 20‐23 dpm. The limits of detection were 7‐8 dpm (one third of the limit of quantification). For blood and plasma samples, dpm/mL was based on measured dpm/g and assuming a density of approximately 1.0 g/mL.
4
Evaluating Proliferative Response in U87MG Cells
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The proliferative response was evaluated in human U87MG cell line by 3H-thymidine incorporation assay [24 (link)]. Briefly, 1 × 106 cells per well were cultured in 12-well plates and incubated with Vc (100 nM) and Et (2 μM) alone or with CD73 inhibitor (AOPCP) or A3AR antagonist (MRS1220) for 24 h. Then 0.5 μCi/mL of 3H-thymidine per well was added for 24 h. After incubation, cells were harvested and the resulting trichloroacetic acid-insoluble materials were collected on 0.45 μm PVDF filters. Once dry, the filters were put into plastic vials with 4 mL of liquid scintillation. Incorporated radioactivity was measured in a liquid scintillation counter (Packard). The results were plotted as counts per minute (cpm) and normalized to the total protein content of each culture well.
5
Quantifying S1P Release from Hydrogels
6
Measurement of Cellular Glucose Uptake
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Glucose uptake was measured according to Kim et al. [24 ] with some modifications [25 (link)]. Briefly, after 10 days of treatment, cells were washed twice in serum-free DMEM and pre-incubated in this medium at 37 °C for 16 h. After this starvation period, cells were washed twice with Krebs–Ringer bicarbonate buffer (KRB) and incubated at 37 °C for 30 min with 100 nM insulin (or not, for the negative control). To initiate glucose uptake, 2-deoxy-[1-3H]-glucose (1 μCi/mL) diluted in 0.1 mMD-glucose solution was added to each well and the plates then incubated at 37 °C for 10 min. After incubation, the cells were washed twice with ice-cold KRB buffer and lysed in 0.1 N NaOH. Half of the content of each well was transferred to scintillation vials, and 10 mL of scintillation cocktail (Ultima Gold, Perkin Elmer, Boston, WalthamMA, USA) was added. The radioactivity incorporated into the cells was measured using a liquid scintillation counter (Hewlett Packard, USA). The BCA protein content was assayed for each point on the remaining half.
7
Biodistribution of 14C-labeled 12-TPP
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A 12-TPP compound labeled with 14C at the alpha-carbon position was purchased from American Radiolabeled Chemicals (St. Louis, MO). Animals bearing A375 xenografts were administered 2.5 μCi of 14C labeled 12-TPP via tail vein injection, oral gavage, intraperitoneal injection, or hydrogel peritumorally. All animals were euthanized via isoflurane overdose or CO2 exposure at 0.5 h, 3 h, 12 h, 24 h, or 48 h post administration of 14C-12-TPP. Organs were harvested, dried, ground, and counted using a liquid scintillation counter (Packard). Activity was normalized to the total activity recovered in the analysis and represented as normalized activity per gram of tissue. N = 2 mice for each route of administration and time point.
8
Caco-2 and T-84 Cells 5-HT Uptake
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Uptake of [3H]5-HT by Caco-2 cells and T-84 cells under unstimulated conditions was measured by assessing Na+- and Cl− dependent [3H]5-HT uptake as previously described by us60 (link). [3H]5-HT uptake was initiated by the addition of 300 μl of uptake buffer containing 25–50 nM [3H]5-HT (Perkin Elmer) for a time period of 5 min (linear range of uptake). The uptake was stopped by washing twice with ice-chilled 1X PBS. The cells were lysed completed by 500 μl 0.5 N NaOH overnight at 4 °C. Radioactivity was measured with a liquid scintillation counter (Packard) and relative protein levels were measured by Bradford (Bio-Rad).
9
Placental Transport Kinetics of Radiolabeled Solutes
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Transport of 3H2O was measured with the use of Ussing chambers (Physiologic Instruments, San Diego, CA, USA) containing 5 mL of oxygenated (95% O2/5% CO2) Krebs buffer as well as physiological concentrations of AAs and glucose, as we described [53 (link), 54 (link)]. Pieces of placental tissue (1 cm2) were mounted onto Ussing chambers, followed by the addition of 3H2O (20 µL), 0.2 mmol/L L-[U-14C]Arg, 0.5 mmol/L L-[U-14C]glutamine, or 1 mmol/L [U-14C]glycine (similar to physiological concentrations of AAs in the pig plasma) to the “mucosal” side of each chamber. The specific radioactivity of 3H2O on the “mucosal” side of the chamber was 500 dpm/µL H2O, whereas the specific radioactivities of 0.2 mmol/L L-[U-14C]Arg, 0.5 mmol/L L-[U-14C]glutamine, and 1 mmol/L [U-14C]glycine on the “mucosal” side of the chamber were 3 × 104, 1.2 × 104, and 6 × 103 dpm/nmol, respectively. Thereafter, an aliquot of 20 µL solution was obtained from the “serosal” side of the chamber at 5, 10 and 15 min for the measurement of 3H2O and 14C radioactivity using a Packard liquid scintillation counter [47 (link)].
10
Insulin-Stimulated Glucose Uptake in Rat Soleus Muscle
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After fasting overnight, rats were anesthetized using urethane (1 g/kg, intraperitoneally) and euthanized; the soleus muscles were obtained by dissection (25-30 mg) and preincubated in 12-well plates at their resting length. The soleus muscles were then incubated in 2 mL of Krebs-Henseleit buffer (KHB) containing 40 mmol/L mannitol, 0.1% bovine serum albumin (BSA) and 8 mmol/L glucose with or without 2 mIU/mL insulin at 37 °C for 2 h. The muscles were then transferred to 12-well plates in 2 mL of KHB containing 40 mmol/L mannitol, 0.1% BSA, 1.5 μCi/ml [3H]-2-deoxy-D-glucose and insulin at the same concentration as was used during the preceding incubation. The plates were placed in a water bath at 37 °C under continuous shaking (60 beats/min) and bubbling of 95% O2 and 5% CO2. After 30 min, the muscles were placed in scintillation vials containing 100 μL of formic acid and 100 μL of 30% hydrogen peroxide and were counted in a Packard liquid scintillation counter with channels preset for simultaneous 3H [13 (link), 14 (link)].
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