For in vivo cholesterol and triglyceride synthesis assays, liver was isolated from mice 1 hour after intraperitoneal injection with 10 mCi of [3H] H2O (Perkin Elmer) in 0.2 mL of phosphate‐buffered saline (PBS). In parallel, trunk blood was also collected, and the [3H] H2O activity in the plasma was measured. For triglyceride detection, the lipid fraction was extracted by homogenizing the liver in chloroform:methanol (2:1). Lipids were separated by thin‐layer chromatography (TLC) (hexane:diethyl ether:glacial acetic acid, 80:20:1). For cholesterol measurements, the liver was saponified before lipid extraction and TLC. The tracer incorporated into the triglyceride or cholesterol fraction was measured by liquid scintillation counting, and synthesis rates were calculated as nmol of 3H‐labeled water incorporated into cholesterol per milligram of protein per hour as described.20
3h h2o
Manufactured by PerkinElmer
Sourced in United States
[3H]-H2O is a radioactively labeled form of water, where the hydrogen atoms are substituted with the radioactive isotope tritium (3H). This product is commonly used as a tracer in various scientific applications, such as biochemical and biological research.
Automatically generated - may contain errors
Lab products found in correlation
9 10 3h palmitic acid, by PerkinElmer (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium without glucose, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
1480 wizard 3, by PerkinElmer (1 mentions)
Lsc 5100, by Hitachi (1 mentions)
Hplc grade acetone, by Merck Group (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin mix, by Thermo Fisher Scientific (1 mentions)
Deadend fluorometric tunel system kit, by Promega (1 mentions)
Liquid scintillation counting cocktail, by PerkinElmer (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
2 deoxy d 1 2 3h glucose, by PerkinElmer (1 mentions)
Scintillation cocktail, by PerkinElmer (1 mentions)
5 protocols using 3h h2o
1
In Vivo Cholesterol and Triglyceride Synthesis
2
Quantifying Cellular Uptake of Radiotracers
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In order to quantify the cell uptake of ions or small molecules after NIR light irradiation 111InCl3, 111In‐DTPA (Nihon Medi‐Physics Co. Ltd., Tokyo, Japan) and 3H‐H2O (PerkinElmer, Inc.) were used. 3T3‐HER2 cells were seeded at 1 × 106 on 35‐mm dishes. Cells in the Tra‐IR700 (+) group were incubated with Tra‐IR700 (5 μg/mL) for 1 hour. After washing with PBS, fresh medium or 2 different high osmotic pressure media containing these radiotracers were added to each dish. The high osmotic pressure media were prepared by adding 25 mmol/L NaCl or 50 mmol/L dextran (MW ~6000) to RPMI‐1640. Immediately after adding medium, cells were exposed to NIR light (2 J/cm2) using the LED. The cells were incubated for 3 minutes or 60 minutes at 37°C after irradiation, and they were then detached from the dishes by incubation with trypsin EDTA. After cell‐washing centrifugation with PBS, the cells were lysed in 0.1 N NaOH and radioactivity was measured with an automatic γ‐counter (1480 Wizard 3; PerkinElmer, Inc.) or a liquid scintillation counter (LSC‐5100; Hitachi‐Aloka Medical, Tokyo, Japan). Protein concentration was measured with the BCA protein assay kit (Thermo Fisher Scientific Inc.). The uptake was presented as percentage dose per milligram of protein.
3
Fatty acid metabolism study
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Cell culture reagents, as RPMI-1640 medium without glucose, fetal bovine serum (FBS) and penicillin/streptomycin mix, were purchased from Invitrogen/Gibco (California, USA). The [9,10-3H]-palmitic acid, [3H]-H2O and liquid scintillation counting solution were from PerkinElmer, (Massachusetts, USA). Essentially fatty acid-free bovine serum albumin (BSA), palmitic acid and HPLC-grade acetone was from Sigma-Aldrich (Madrid, Spain). Recombinant human IL-6 was acquired from Peprotech (London, UK).
4
Cell Culture and Radiotracer Assays for Placental Metabolism
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Cell culture reagents (RPMI-1640 medium without glucose and fetal bovine serum) were purchased from Gibco, California, USA. The [9,10-3H]-palmitic acid, [3H]-H2O, 2-[1,2-3H]-deoxy-D-glucose, [1-14C]-mannitol, and liquid scintillation counting cocktail were from Perkin Elmer, Massachusetts, USA. Hydrocortisone sodium phosphate, as GC treatment used in this study, was purchased from Nycomed Pharma (Zurich, Switzerland). Lipoprotein lipase (LPL) was purchased from Sigma-Aldrich (St. Louis, USA). MTT for the colorimetric assay for the nonradioactive quantification of cellular metabolic activity was purchased from Roche (Mannheim, Germany). Apoptosis in the placental explants was analyzed by the Dead-End TM Fluorometric TUNEL System Kit (Promega, Madison, WI, USA) and the Caspase-GLO 3/7 Assay Kit (Promega, Madison, USA).
5
Estimating Intracellular Water Volume in BeWo and JEG-3 Cells
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To investigate intracellular concentration of GBP, intracellular water volumes of BeWo and JEG-3 cells were estimated using modified methods described by Rottenberg et al. [21] and Shiraya et al. [22] . BeWo cells and JEG-3 cells were seeded on 24-well collagen-coated plastic plates. After removal of growth medium, cells were washed with a transport buffer and pre-incubated with 0.5 mL of buffer. The buffer was removed and 0.5 mL of buffer containing [ 3 H]-H 2 O (1 µCi/mL) (PerkinElmer, Boston, MA), and [ 14 C]inulin-carboxyl (0.5 µCi/mL) (American Radiolabeled Chemicals, Inc. St. Louis, MO) was added to cells. The cells were incubated in the buffer for 10 min. The extracellular buffer was collected, and cells were solubilized with 1 % sodium dodecyl sulfate (SDS)/0.2 N NaOH. The samples were mixed with 3 mL of scintillation cocktail (PerkinElmer) to measure radioactivity using a liquid scintillation counter.
The intracellular water volume was calculated using the following equation:
where V s is the volume of extracellular buffer, 3 H c is the 3 H counts in cells, 3 H s is the 3 H counts in supernatant, 14 C c is the 14 C counts in cells, and 14 C s is the 14 C counts in supernatant.
The intracellular volume was expressed relative to protein.
The intracellular water volume was calculated using the following equation:
where V s is the volume of extracellular buffer, 3 H c is the 3 H counts in cells, 3 H s is the 3 H counts in supernatant, 14 C c is the 14 C counts in cells, and 14 C s is the 14 C counts in supernatant.
The intracellular volume was expressed relative to protein.
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