The culture medium was removed and the cells were preincubated with the transport buffer (Hanks’ balanced salt solution containing 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0) at 37 °C for 15 min. The uptake assays were initiated by replacing the medium with transport buffer containing 135 nM [3H]glycylsarcosine at 37 °C in the presence or absence of 3 mM ibuprofen (Wako Pure Chemical Industries) (PEPT1 inhibitor) or at 4 °C. Uptake was stopped by the addition of ice-cold transport buffer; the cells were rinsed using the same buffer. The cells were lysed with 0.2 M NaOH solution (0.5 mL/well) containing 0.5% sodium dodecyl sulfate (SDS). Radioactivity was measured through liquid scintillation counting, using 3 mL Clear-sol I (Nacalai Tesque, Inc., Kyoto, Japan) as the scintillation fluid. The total protein in differentiated cells was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
Clear sol 1
Manufactured by Nacalai Tesque
Sourced in Japan, United States
Clear-sol I is a solvent used for the preparation of samples in various laboratory applications. It is a colorless, transparent liquid with a specific gravity of approximately 1.01 g/cm³. Clear-sol I is designed to provide effective solvent properties for the dissolution and handling of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ibuprofen, by Fujifilm (1 mentions)
Trichloroacetic acid tca, by Fujifilm (1 mentions)
Methyl 3h thymidine, by American Radiolabeled Chemicals (1 mentions)
Kaleidagraph 4, by Synergy Software (1 mentions)
Lsc 8000, by Aloka (1 mentions)
Dr2800, by HACH (1 mentions)
20a hplc, by Shimadzu (1 mentions)
Cdd 10avp, by Shimadzu (1 mentions)
Shodex asahipak nh2p 50 4d anion column, by Resonac (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Shim pack scr 102h column, by Shimadzu (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Deuterium oxide, by Merck Group (1 mentions)
Toluene, by Fujifilm (1 mentions)
L lactide, by Tokyo Chemical Industry (1 mentions)
Soluene 350, by PerkinElmer (1 mentions)
3h l leucine, by PerkinElmer (1 mentions)
Dry bath incubator, by Nippon Genetics (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
8 protocols using clear sol 1
1
Probing PEPT1-Mediated Glycylsarcosine Uptake
2
Docetaxel Uptake in MRP2-Expressing Cells
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MRP2-expressing and vector-transfected MDCKII cells were seeded in 12-well plates at 1.25 × 105 cells/well. After 48-h incubation, the medium was replaced with fresh medium containing [3H]-docetaxel and the cells were incubated for 24 h. The cells were then washed three times with 1 ml of ice-cold phosphate-buffered saline, solubilized with 500 µl of 0.2 N NaOH and stored overnight at 4 °C. After 250 µl of 0.4 N HCl was added to solubilized cells, aliquots (600 µl) were transferred to scintillation vials. The radioactivity associated with the cells and the incubation buffer was measured with a liquid scintillation counter after the addition of 3 ml of scintillation fluid (Clear-sol I; Nacalai Tesque, Kyoto, Japan) to the scintillation vials.
3
LLNA Assay for Skin Sensitization
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LLNA was conducted mainly in accordance with OECD TG No. 429. Groups of BALB/c female mice (8 weeks of age) were treated topically on the dorsum of ears with 25 µL of BGP at 4 concentrations in 2-fold serial dilutions or with an equal volume of vehicle alone (AOO). Treatment was performed daily for 3 consecutive days (days 0, 1, and 2). On day 5, all mice were injected through the tail vein with 0.25 mL [methyl-3H]-thymidine (20 μCi; American Radiolabeled Chemicals, St. Louis, MO, USA). After 5 h, mice were euthanized by a lethal overdose of isoflurane, the auricular lymph nodes, as the draining lymph nodes, were excised and lymph node cells were collected individually. Each lymph node cell suspension was washed with phosphate-buffered saline, pelleted by centrifugation, and then resuspended in 5 mL of 5% (v/v) trichloroacetic acid (TCA) (Fujifilm Wako) and precipitated at 4 °C for 18 h. After centrifugation of the cell solution, pellets were resuspended in 1 mL of 5% TCA, and each sample was mixed with 4 mL of scintillation cocktail Clear-sol I (Nacalai Tesque). Incorporation of [methyl-3H]-thymidine was measured by β-scintillation counting and expressed as disintegrations per minute (DPM) per mouse.
4
Kinetic Analysis of Uric Acid Uptake Inhibition
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[ 14 C]UA was measured for radioactivity using a liquid scintillation counter (LSC8000; Aloka, Tokyo, Japan) after adding a liquid scintillation cocktail (Clear-sol I; Nacalai Tesque, Kyoto, Japan) to each sample. The inhibitory effects of the drugs on UA uptake are presented as percentages of the control, and kinetic parameters for the inhibitory effect were estimated using non-linear least-squares regression analysis in KaleidaGraph 4.0 (Synergy Software, Reading, PA, USA), according to the following equation:
% of control = 100 × IC 50 /(IC 50 + I)
where IC 50 and I are the half-maximum inhibitory concentration and the concentration of the inhibitor used, respectively.
% of control = 100 × IC 50 /(IC 50 + I)
where IC 50 and I are the half-maximum inhibitory concentration and the concentration of the inhibitor used, respectively.
5
Quantitative Analysis of Microbial Metabolites
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Ammonium concentrations were determined using Nessler’s method with a UV-visible spectrophotometer (DR-2800; Hach, Loveland, CO, USA). Nitrite and nitrate concentrations were determined using ion chromatography (HPLC 20A; Shimadzu, Kyoto, Japan) with a Shodex Asahipak NH2P-50 4D anion column (Showa Denko, Tokyo, Japan) and UV-VIS detector (SPD-20A; Shimadzu) following the filtration of samples through 0.2-μm pore size membranes (Advantec, Tokyo, Japan), as previously described (14 (link)). The concentration of VFAs was determined using HPLC (HPLC 20A; Shimadzu) with a Shim-packSCR-102H column (Shimadzu) and conductivity detector (CDD-10A vp; Shimadzu) following the filtration of samples through 0.2-μm pore size membranes (Advantec). [14C]bicarbonate uptake was confirmed by liquid scintillation counting. The biomass was collected, washed three times with phosphate buffered saline, and mixed with the scintillation cocktail (Clear-sol I; Nacalai Tesque, Kyoto, Japan). Radioactivity was subsequently determined using an LSC-5100 liquid scintillation counter (Hitachi-Aloka Medical, Tokyo, Japan).
6
Synthesis and Characterization of Radiolabeled Polymeric Drug Delivery System
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Methoxy-polyethylene glycol (mPEG, 5kDa), deuterium chloroform, and deuterium oxide were purchased from Sigma Aldrich (St. Louis, MO, USA). l- lactide and tin (II) 2-ethylhexanoate (Sn (Oct)2) were purchased from Tokyo Chemical Industry (Tokyo, Japan). d- lactide was purchased from Leap Labchem (Hangzhou, China). Tamoxifen-free base (TAM) was purchased from MP Biomedicals (Santa Ana, CA, USA). Dichloromethane (DCM), diethyl ether, toluene, dimethyl sulfoxide, and N, N-dimethyl formamide (DMF, HPLC grade) were purchased from Fujifilm Wako Chemical Industries (Tokyo, Japan). Acetonitrile (MeCN, HPLC grade) and LiBr were purchased from Kanto Chemicals (Tokyo, Japan). Soluene 350 was purchased from Perkin-Elmer (Waltham, MA, USA). Clear-Sol I was purchased from Nacalai Tesque (Boston, MA, USA). 14C-TAM [N-methyl-14C] was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). 111Indium chloride ([111In] InCl3) was supplied by Nihon Medi-Physics Co. (Tokyo, Japan). All other chemicals were obtained commercially as reagent-grade products.
7
HeLa Cell Leucine Uptake Assay
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HeLa cells were seeded in 24-well plates at a density of 1.0 × 105 cells per well, in 1 mL of medium. After overnight incubation, the plates were placed on the dry bath incubator (Nippon Genetics Europe, Dueren, Germany) heating at 34 °C, 37 °C, or 40 °C. After removal of the medium, the cells were rinsed with warmed buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM d -(+)-Glucose, 1.2 mM CaCl2·H2O, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 25 mM HEPES), and pre-incubated with warmed buffer for 10 min. The cells were incubated with 1 µCi/mL l -[3H]-leucine (Moravek Biochemicals, Richland, WA, USA) and polymer or BCH (Sigma-Aldrich) (2 mM) as the inhibitor, for 10 min. The cells were rinsed with cooled buffer and added to 0.1 M NaOH (250 μL) at 4 °C overnight. Following the overnight incubation, 0.1 M HCl (250 μL) was added to each well, and the sample solutions (400 μL) were dissolved in liquid scintillation solvent (Clear-sol I, Nacalai Tesque, Kyoto, Japan) in a vial. l -[3H]-Leucine was detected using a Packard Tri-Carb 3170 TR/SL liquid scintillation analyzer (PerkinElmer Japan, Kanagawa, Japan). The protein concentrations in the samples were determined using a Pierce BCA Protein Assay Reagent Kit.
8
HSD17b1 Activity Quantification by TLC
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HSD17b1 activity was measured using thin layer chromatography, as described previously (Kitawaki et al. 2000) (link). Briefly, cells were washed twice with phenol-red-free DMEM/Ham's F-12, and then incubated at 37 8C/5% CO 2 for 6 h with 0.5 ml of serum-free medium containing [6,7-3 H]estrone (Perkin Elmer, Waltham, MA, USA) (1.8!10 6 dpm, 37 mM). The reaction was stopped by transferring the medium to the test tubes containing 2 ml chloroform and the corresponding carrier steroids: [4-14 C]estradiol (Perkin Elmer) (1.3!10 4 dpm) and nonradioactive estrone and estradiol (0.2 mg each). The steroids were isolated by thin-layer chromatography using Silicagel 60 F254 (0.25 mm; Merck) in a system of chloroform:ethyl acetate (4:1, v/v). The aliquot was mixed with Clear-sol I (Nacalai Tesque), and radioactivity was measured using a scintillation counter (Beckman Coulter, Fullerton, CA, USA). Enzyme activity was calculated and normalized according to the ratios of the estradiol formed. Protein concentration (pmol/mg protein per h) was measured by the Bradford method.
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