Cells were harvested by trypsinization and washed once in phosphate-buffered saline (PBS). Cells were resuspended gently in ∼100 μl of PBS, and 75 mM potassium chloride (Kanto Chemical, Tokyo, Japan) was added slowly up to a volume of 10 ml. The samples were then incubated for 10 min in a 37°C water bath. Following that, 1 ml of Carnoy solution (3:1 methanol:acetic acid) was added to each sample, and samples were centrifuged for 10 min at 1000 rpm. Supernatant was discarded and cells were gently resuspended in a small amount of the remaining supernatant. Carnoy solution (10 ml) was added slowly to the samples and cells were fixed for at least 1 h at room temperature (RT). Samples were centrifuged for 10 min at 1000 rpm, supernatant was discarded, and cells were fixed again with 10 ml of fresh Carnoy for at least 1 h at RT. Samples were then centrifuged and pellets were resuspended in a suitable volume (∼0.5–1.5 ml) of Carnoy solution. A sample of 20 μl of this suspension was drawn up and dropped from a height (10–30 cm) onto glass slides and allowed to air-dry. DNA was then stained with Giemsa (Life Technologies, Thermo Fisher Scientific).
Potassium chloride (kcl)
Manufactured by Kanto Chemical
Sourced in Japan
Potassium chloride is a white, crystalline chemical compound with the formula KCl. It is a common laboratory reagent used in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Kanto Chemical (4 mentions)
Sodium chloride (nacl), by Fujifilm (2 mentions)
Potassium dihydrogen phosphate, by Fujifilm (2 mentions)
Sodium hydroxide (naoh), by Kanto Chemical (2 mentions)
Tetrahydrofuran thf, by Merck Group (2 mentions)
Giemsa, by Thermo Fisher Scientific (1 mentions)
Potassium chloride (kcl), by Fujifilm (1 mentions)
Disodium phosphate, by Fujifilm (1 mentions)
Potassium dihydrogen phosphate, by Kanto Chemical (1 mentions)
Sodium dihydrogen phosphate, by Kanto Chemical (1 mentions)
Acetic acid, by Kanto Chemical (1 mentions)
Sodium acetate, by Kanto Chemical (1 mentions)
Calcium chloride, by Fujifilm (1 mentions)
Polyethylene glycol monomethyl ether, by Tokyo Chemical Industry (1 mentions)
Cyclophosphamide monohydrate, by Merck Group (1 mentions)
Glacial acetic acid, by Beijing Chemical Works (1 mentions)
Absolute methanol, by Beijing Chemical Works (1 mentions)
Sucrose, by Sinopharm (1 mentions)
Agar powder, by Fujifilm (1 mentions)
Yeast extract, by Fujifilm (1 mentions)
11 protocols using potassium chloride (kcl)
1
Giemsa Staining of Chromosomes
2
pH Adjustment with Phosphate Buffers
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The pH was adjusted by adding disodium phosphate (10 mM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing sodium chloride (137 mM, FUJIFILM Wako Pure Chemical Corporation) and potassium chloride (2.68 mM, KANTO CHEMICAL, Tokyo, Japan) to potassium dihydrogen phosphate (2.0 mM) containing sodium chloride (137 mM, FUJIFILM Wako Pure Chemical Corporation) and potassium chloride (2.68 mM, FUJIFILM Wako Pure Chemical Corporation).
3
Cellulase-Assisted Hydrogel Synthesis
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CMC (molecular weight = 8–10 × 104 as specified by manufacturer) with a 0.70 degree of substitution was obtained from Daicel Co., (Osaka, Japan). EGDE was purchased from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan). Liquid paraffin (Moresco white P-100, 19.04 mm2/s) was purchased from Moresco Co., (Kobe, Japan). Sodium hydroxide was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Methanol was purchased from Godo Co., Ltd. (Tokyo, Japan). The SPA was supplied by S. T. Co., (Tokyo, Japan). Trichoderma viride cellulase ONOZUKA R-10 was purchased from Yakult Pharmaceutical Co., Ltd. (Tokyo, Japan). The PBS and sodium acetate buffer used were analytical grade chemicals, i.e., sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, and acetic acid, purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).
4
Synthesis of PEG-based Polymer Compounds
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2-1. Material 2,2′-Azobisisobutyronitrile (AIBN) (98%), calcium chloride (95%), and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (99%) were purchased from FUJIFILM Wako Pure Chemical Corporation. Osmium tetroxide (4% in water) and polyethylene glycol monomethyl ether (Mw: 400, 2000, and 4000) were purchased from Tokyo Chemical Industry. Sodium chloride (99%), magnesium chloride hexahydrate (99%), potassium chloride (99.5%), and dry N,N-dimethylformamide (DMF) (99.5%) were purchased from Kanto Chemical. Dimethyl sulfoxide (DMSO, 98%) was purchased from Kishida.
5
Cytogenetic Analysis of Cell Lines
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Cyclophosphamide monohydrate (Sigma-Aldrich, St Louis, U.S.), colchicine, purity ≥96.0% (Fluka, Buchs, Switzerland), sucrose (Sinopharm Chemical Reagent Co., Ltd, Beijing, China), sodium chloride injection (Shijiazhuang No.4 Pharmaceutical Co., Ltd, Shijiazhuang, China), potassium chloride (Kanto Chemicalco. Inc, Kagaku, Japan), absolute methanol (Beijing Chemical Works, Beijing, China), glacial acetic acid (Beijing Chemical Works, Beijing, China), Giemsa and acridine orange staining buffer (Sinopharm Chemical Reagent Co., Ltd, Beijing, China).
6
Recombinant Protein Production in E. coli
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BP1 was produced by a fermentation process using E.coli, which was previously reported in the literature28 ,44 and provided in an unprocessed powder form by Spiber Inc. The amino acid composition of BP1 was alanine (16.4%), tyrosine (12.0%), glutamine (23.7%), glycine (21.9%), proline (15.3%), serine (9.3%) and others (1.4%). The protein sequence structure is shown in Fig. 1 .
KH2PO4, K2HPO4, NaCl, Na2HPO4·H2O, NH4Cl, MgCl2·6H2O, CaCl2, FeCl3·6H2O, Yeast extract, and agar powder were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Na2SO4, KCl, HCl, Na2SO4, NaHCO3, and NaOH were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Plysurf was purchased from DKS Co. Ltd. (Kyoto, Japan). All chemicals were of reagent grade and used without further purification. Pronase E (P5147 Protease Type XIV from Streptomyces griseus) was purchased from Sigma Aldrich, Proteinase K was purchased from TaKaRa Bio Inc (Kusatsu, Japan), and chymotrypsin was purchased from NACALAI TESQUE, INC (Kyoto, Japan).
KH2PO4, K2HPO4, NaCl, Na2HPO4·H2O, NH4Cl, MgCl2·6H2O, CaCl2, FeCl3·6H2O, Yeast extract, and agar powder were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Na2SO4, KCl, HCl, Na2SO4, NaHCO3, and NaOH were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Plysurf was purchased from DKS Co. Ltd. (Kyoto, Japan). All chemicals were of reagent grade and used without further purification. Pronase E (P5147 Protease Type XIV from Streptomyces griseus) was purchased from Sigma Aldrich, Proteinase K was purchased from TaKaRa Bio Inc (Kusatsu, Japan), and chymotrypsin was purchased from NACALAI TESQUE, INC (Kyoto, Japan).
7
Heat Shock and Stress Response Protocol
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Prior to stress treatment, the cells were grown to mid-log phase at 27 °C. Heat shock was imposed by transferring the culture tubes to a water bath at 42 °C for the indicated time. To the culture medium, 100 mM sodium arsenate dibasic heptahydrate (Katayama Chemical Industries Co., Ltd., Osaka, Japan) stock solution, 5.0 M KCl (Kanto Chemical Co., Inc., Tokyo, Japan, Cat. No. 32326-00) stock solution, Sorbitol (Kishida Chemical Co., Ltd., Osaka, Japan, Cat. No. 000-73236) and Hydrogen peroxide solution with concentration of 30% (wt/vol) (Santoku Chemical Industries Co. Ltd., Tokyo, Japan, CAS No. 7722-84-1) were added at the indicated concentrations. For glucose deprivation, cells were collected by centrifugation, washed in media lacking glucose and briefly centrifuged. Cells were then re-suspended in media lacking glucose and incubated at 27 °C for the indicated time.
8
Purification and Analysis of RSPO1 Protein
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Cells were washed twice with phosphate-buffered saline [PBS; containing 137 mM NaCl, 2.7 mM KCl (Kanto Chemical Co., Inc.), 10 mM Na2HPO4 and 1.8 mM KH2PO4] and cultured in serum-free DMEM with 50 µg/ml heparin sodium salt (catalogue no. H3149; Sigma-Aldrich) for 24 h. Conditioned medium (CM) was then collected, and Ni-nitrilotriacetic acid (NTA) agarose (Qiagen GmbH, Hilden, Germany) was added to the CM, and the mixture was incubated for 2 h at 4°C. Next, the Ni-NTA agarose was washed with washing buffer A [900 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 20 mM imidazole (USB Corporation, Cleveland, OH, USA)], and Ni-NTA-bound RSPO1 was subsequently eluted with an elution buffer (900 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 500 mM imidazole, pH 7.4). Upon concentration with Ni-NTA agarose, the total amount of protein in the CM was estimated from the total amount of protein in the cell lysates. Samples were next electrophoresed and analyzed by western blotting as mentioned above.
9
Molecular Extraction and Analysis Protocol
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Tokyo Chemical Industry Co., Ltd. (Tokyo) supplied TBHP (CAS: 75-91-2, purity: 70.7%) P-gal (phenylβ-d-galactoside) (CAS: 2818-58-8). Kanto Chemical Co., Inc. supplied disodium hydrogenphosphate (CAS: 10039-32-4), NaCl (CAS: 7647-14-5), KCl (CAS: 7447-40-7), and sucrose. N-ethyl-N-nitrosourea (ENU), a positive control substance, was purchased from Toronto Research Chemicals Inc. (Ontario, Canada). Next, 0.5 w/v% MC400 solution, Proteinase K, SDS, and Potassium Dihydrogen Phosphate were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka). EDTA and RNase A were purchased from Nippon Gene Co., Ltd. (Tokyo, Japan).
10
Lipid/Polymer Membrane Fabrication and Characterization
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The lipid used for making lipid/polymer membrane was tetradodecylammonium bromide (TDAB), and the preparation solvent was tetrahydrofuran (THF), both of which were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dioctyl phenyl-phosphonate (DOPP) and polyvinyl chloride (PVC) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The caffeine sample was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The modifiers studied were 2,6–DHBA, 3,4-dihydroxybenzoic acid (3,4–DHBA), 3,5–dihydroxybenzoic acid (3,5–DHBA), 2–hydroxybenzoic acid (2–HBA), 2,5–dihydroxybenzoic acid (2,5–DHBA), and 2,4,6–trihydroxy benzoic acid (2,4,6–THBA). All modifiers were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Tannic acid and potassium chloride (KCl) were used to make the reference solution, and these were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Figure 1 shows the molecular formulae of TDAB, DOPP, and caffeine.
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