Mice (12 h fasted, n = 6–7/genotype) were injected retro-orbitally with 100 µL PBS containing 2 µCi of [3H]oleic acid (Perkin Elmer) and perfused with 10 mL PBS through the left ventricle 5 min after injection. Excised tissues were homogenized in 500 µL PBS and aliquots (100 µL) added to 5 mL scintillation fluid (Optiphase HiSafe 3, Perkin Elmer) for radioactivity measurement. Tissue FA uptake was adjusted by tissue weight and by antrum radioactivity which did not differ between groups.
Optiphase hisafe 3
Manufactured by PerkinElmer
Sourced in United States, Sweden
The Optiphase HiSafe 3 is a liquid scintillation counting cocktail designed for high-efficiency counting of various radioactive samples. It provides a high luminescence yield and low background count rates for accurate measurement of radioactive samples.
Automatically generated - may contain errors
Lab products found in correlation
D 2 3h glucose, by PerkinElmer (2 mentions)
Gstf filter, by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Prism 5, by GraphPad (2 mentions)
3h oleic acid, by PerkinElmer (1 mentions)
Filter plate, by Merck Group (1 mentions)
Desipramine hydrochloride, by Merck Group (1 mentions)
Orcein, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Masson s trichrome, by Merck Group (1 mentions)
Haematoxylin eosin, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Entellan, by Merck Group (1 mentions)
Trilux beta counter, by PerkinElmer (1 mentions)
Advanced protein assay reagent, by Cytoskeleton (1 mentions)
2 deoxy d 1 14c glucose, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Microbeta jet, by PerkinElmer (1 mentions)
Biomax mr 1 film, by Kodak (1 mentions)
Kh232po4, by PerkinElmer (1 mentions)
45 protocols using optiphase hisafe 3
1
Measuring Tissue Fatty Acid Uptake
2
Measuring Oleate and Palmitate Uptake In Vivo
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Oleate and palmitate uptake in vivo was measured as previously described (Hagberg et al, 2010 (link)). Briefly, adult rats were administered 14C-oleate or 14C-palmitate dissolved in olive oil by oral gavage and tissues were collected after 24 h. In another experiment, 3H-2-deoxy-glucose or 14C-oleate was administered via a tail vein, and hearts and blood were collected 30 min later. Tissues were lysed and radioactivity was measured from serum and lysates by liquid scintillation using Optiphase HiSafe 3 (Perkin-Elmer) and Wallac LS Counter (Turku, Finland).
3
Autoantibody Detection Protocol
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Immunoprecipitation of radiolabeled antigens was used to screen sera for autoantibodies against tryptophan hydroxylase-1. Human complementary DNA from the antigen in expression vector was used to perform the assay. In vitro transcription and translation were performed in the presence of 35S-methionine, according to the manufacturer’s protocol (Promega TNT Systems). Immunoprecipitation was performed in 96-well plates overnight at 4°C at 300 rpm with serum samples (2.5 µl) and 30,000 cpm of radiolabeled protein. A positive control (positive patient serum to each antigen) and a negative control, 4% bovine serum albumin, were included in each plate. All samples were analyzed in duplicates. The immune reaction was transferred to filter plates (Millipore) and immune complexes were captured to Protein A sepharose (nProtein A Sepharose 4 fast flow, GE Healthcare) during a 45 min incubation at 4°C at 300 rpm. After ten washing steps with wash buffer (150mM NaCl, 20 mM TrisHCl pH 8, 0.15% Tween 20, and 0.1% BSA), plates were dried and scintillation fluid (Optiphase, HiSafe 3, PerkinElmer) was added. Radioactivity was then measured in a beta counter (1450 Microbeta Trilux, Wallac). Autoantibody index values were calculated according to the following: (sample value – negative control value)/(positive control value – negative control value) x 100.
4
Noradrenaline Uptake Quantification Protocol
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The following drugs were used: levo-[ring-2,5,6-3H]-noradrenaline, specific activity 44.8 Ci/mmol (DuPont NEN, I.L.C., Lisboa, Portugal); the scintillation mixture used was from OptiPhase ‘Hisafe’ 3, PerkinElmer, I.L.C. (Lisboa, Portugal); desipramine hydrochloride purchased from Sigma-Aldrich (Sintra, Portugal). Entellan (mounting medium), Orcein, Masson’s trichrome and haematoxylin/eosin from Merck (Darmstadt, Germany). The following antibodies were used: mouse monoclonal anti-tyrosine hydroxylase antibody (ab137869), from Abcam, London, UK) and Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody, highly cross-adsorbed (Invitrogen, Life Technologies, SA, Madrid, Spain); vectashield mounting medium with DAPI (Vector Laboratories, London, UK). Stock solutions were made up in ultrapure water and diluted in superfusion medium immediately before use.
5
Transporter Function Assay in Oocytes
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Oocytes were injected with RNA-encoding transporters or an equivalent volume of water as a negative control. A minimum of 3 days after injection, oocytes were incubated for 10 min in uptake solutions containing 3H-l -substrates (PerkinElmer) ± GPNA where specified, in ND96. Oocytes were subsequently rinsed three times in ice-cold ND96. Cells were then lysed in 1 M NaOH and 1% SDS. 3H-l -substrate uptake was measured by scintillation counting using OptiPhase HiSafe 3 (PerkinElmer) and a Trilux beta counter (PerkinElmer). Raw data from each oocyte were normalized to the mean response from the corresponding positive control (all normalized datapoints shown). Inhibition–response data generated using uptake were fit as detailed previously.
6
Quantifying Insulin-Stimulated Glucose Uptake
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For the glucose uptake studies, the 24-hour corticosterone- and/or insulin-treated cells were washed with PBS and stimulated with 0, 20, or 100 nM insulin in 0.1% FF-BSA (03117057001, Roche Diagnostics) in PBS for 15 minutes. Next, 0.05 µCi of 2-[1-14C]-deoxy-d -glucose (PerkinElmer, Waltham, MA) in 0.1% FF-BSA was added to the medium. After an additional 5-minute incubation, the cells were washed twice with cold PBS, lysed with 0.2% SDS solution (Merck, Hohenbrunn, Germany), and protein content in cell lysates was quantified using Advanced protein assay reagent (Cytoskeleton, Denver, CO). Cell lysates were transferred to a scintillation glass vial and homogenized in a scintillation cocktail (Optiphase HiSafe 3, PerkinElmer Health Sciences, Groningen, Netherlands). Radioactivity was detected with a liquid scintillation analyzer (Tri-Carb 2910TR, Packard, PerkinElmer) and reported in counts per minute normalized to protein content.
7
Radiolabeled Alanine Chromatography
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Chromatographic acid alumina and DAPI were obtained from Sigma Chemical Co (St.
Louis, MO, USA). [ 14 C]-Alanine (55 mCi/mmol) was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, USA). Optiphase "Hisafe" 3 was from Perkin Elmer. All the solvents used were of the highest purity available.
Louis, MO, USA). [ 14 C]-Alanine (55 mCi/mmol) was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, USA). Optiphase "Hisafe" 3 was from Perkin Elmer. All the solvents used were of the highest purity available.
8
Radiolabeled Substrate Uptake Assay
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All counterflow assays were performed at 25 °C. For each assay, an aliquot of 2 μl proteoliposomes was added into 98 μl KPM 6.5 buffer plus 1 μCi D-[2-3H] glucose (specific radioactivity 23.4 Ci/mmol, PerkinElmer) or D-[3H] xylose (20 Ci/mmol, American Radiolabeled Chemicals, Inc.). The final concentrations of the external D-[2-3H] glucose and D-[3H] xylose were 0.42 μM and 0.5 μM, respectively. Uptake of radio-labeled substrates was terminated at 30 s by rapidly filtering the solution through 0.22 μm GSTF filters (Millipore) and washed with 2.5 ml KPM 6.5 buffer. The filter was solubilized with 0.5 ml Optiphase HISAFE 3 (PerkinElmer) for 30 min for liquid scintillation counting with MicroBeta JET (PerkinElmer). Liposome without protein was used as negative control.
For inhibition assays, an aliquot of 2 μL proteoliposome pre-incubated with 100 μM indicated inhibitors for 0.5 h on ice was added to 98 μL KPM buffer containing 1 μCi D-[2-3H]-glucose or D-[3H] xylose with 100 μM inhibitors. To determine the IC50 of the inhibitor, a series of concentrations of the inhibitor was applied as indicated.
All experiments were repeated at least three times. Data for liposome-based counterflow assay were analyzed using GraphPad Prism 6.
For inhibition assays, an aliquot of 2 μL proteoliposome pre-incubated with 100 μM indicated inhibitors for 0.5 h on ice was added to 98 μL KPM buffer containing 1 μCi D-[2-3H]-glucose or D-[3H] xylose with 100 μM inhibitors. To determine the IC50 of the inhibitor, a series of concentrations of the inhibitor was applied as indicated.
All experiments were repeated at least three times. Data for liposome-based counterflow assay were analyzed using GraphPad Prism 6.
9
Quantification of Radioactive Samples
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Aqueous and ethanolic solutions were assayed for 14C by scintillation counting in 10 volumes of OptiPhase HiSafe 3 (PerkinElmer, Inc.) aqueous-miscible scintillant.
Gel electrophoretograms and TLCs were dried, then exposed to Kodak BioMax MR-1 film in the dark for ~4 weeks. Dry strips cut from paper chromatograms and paper electrophoretograms, and spots (localized by autoradiography) excised from TLCs, were assayed for 14C by scintillation counting in 2 mL of an aqueous-immiscible scintillant (Gold Star; Meridian Biotechnologies Ltd). Radioactive spots excised from gel electrophoretograms were hydrolysed in 2m trifluoroacetic acid at 100 °C for 1 h, then assayed for 14C by scintillation counting in 10 volumes of aqueous-miscible scintillant.
Gel electrophoretograms and TLCs were dried, then exposed to Kodak BioMax MR-1 film in the dark for ~4 weeks. Dry strips cut from paper chromatograms and paper electrophoretograms, and spots (localized by autoradiography) excised from TLCs, were assayed for 14C by scintillation counting in 2 mL of an aqueous-immiscible scintillant (Gold Star; Meridian Biotechnologies Ltd). Radioactive spots excised from gel electrophoretograms were hydrolysed in 2
10
Evaluation of Liquid Scintillators for Proton Dosimetry
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