Dried D. involucrata leaves were kindly provided by Dr. T. Yamaura, Yamashina Botanical Research Institute (Nippon Shinyaku Co. Ltd., Kyoto, Japan). Repandusinic acid A (6 ) was prepared from geraniin [30 ]. Toyopearl HW-40 (coarse and fine grades; Tosoh Corporation, Tokyo, Japan), YMC-gel ODS-A (S, 75 µm; YMC), Sepabeads SP700, Dia-ion HP-20, and MCI-gel CHP-20P (Mitsubishi Chemical, Tokyo, Japan) were used for column chromatography. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from GIBCO (BRL, Grand Island, NY, USA). Fetal bovine serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Human normal oral cells [(gingival fibroblast (HGF), periodontal ligament fibroblast (HPLF), and pulp cells (HPC)) were established from the first premolar tooth extracted from the lower jaw of a 12-year-old girl [31 (link)]. Human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) were purchased from Riken Cell Bank (Tsukuba, Japan).
Hsc 3
Manufactured by RIKEN Cell Bank
Sourced in Japan
The HSC-3 is a laboratory equipment designed for cryogenic storage and preservation of biological samples, such as cells, tissues, or other biomaterials. The core function of the HSC-3 is to provide a controlled, low-temperature environment to maintain the viability and integrity of the stored samples.
Automatically generated - may contain errors
Lab products found in correlation
Hsc 2, by RIKEN Cell Bank (9 mentions)
Hsc 4, by RIKEN Cell Bank (7 mentions)
Ca9 22, by RIKEN Cell Bank (7 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Diaion hp 20, by Mitsubishi (1 mentions)
Mci gel chp 20p, by Mitsubishi (1 mentions)
Toyopearl hw 40, by Tosoh (1 mentions)
Sodium dichloroacetate, by Merck Group (1 mentions)
Hek293t, by RIKEN Cell Bank (1 mentions)
Mrc 5, by RIKEN Cell Bank (1 mentions)
Melphalan, by Merck Group (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Dms114, by American Type Culture Collection (1 mentions)
Hsc 3, by Thermo Fisher Scientific (1 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
11 protocols using hsc 3
1
Isolation and Characterization of Repandusinic Acid A from Drosera involucrata
2
Cell Culture of HNSCC and HaCaT
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The HNSCC cell lines HSC-2 and HSC-3 were obtained from Riken Cell Bank (Ibaraki, Japan). The human immortalized non-tumorigenic keratinocyte cell line HaCaT was supplied by DKFZ (Heidelberg, Germany). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Japan Ltd.) supplemented with 10% FBS (Life Technologies Japan Ltd.) and antibiotics [penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (25 μg/ml)] at 37°C in a humidified atmosphere containing 5% CO2.
3
Culturing Human Oral Cell Lines
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Human oral normal cells, gingival fibroblast (HGF), periodontal ligament fibroblast (HPLF) and pulp cell (HPC), established from the first premolar tooth extracted from the lower jaw of a 12-year-old girl [13] (link), and OSCC cell lines (Ca9-22, HSC-2, HSC-3, HSC-4), purchased from Riken Cell Bank, Tsukuba, Japan were cultured at 37 °C in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO2 atmosphere. Human oral keratinocytes (HOK) (purchased from COSMO BIO Co./Ltd., Tokyo) was cultured in keratinocyte growth supplement (OKGS, Cat, No. 2652). Primary human gingival epithelial cells (HGEP) (purchased from CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) was growing in CnT-PR medium. Cells were then harvested by treatment with 0.25% trypsin-0.025% EDTA-2Na in PBS(⿿) and either subcultured or used for experiments.
4
Evaluating Cytotoxicity of Compounds in Oral Cancer Cells
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The target cells used in our study were human oral squamous cell carcinoma cell lines Ca9-22 (purchased from RIKEN Cell Bank, Tukuba, RCB-1976), HSC-2 (RCB1945), HSC-3 (RCB1975), HSC-4 (RCB1902), and three human normal oral cells, gingival fibroblast (HGF), periodontal ligament fibroblast (HPLF), and pulp cells (HPC) [established from the first premolar extracted tooth in the lower jaw (because of dysfunctional position or orthodontic treatment) and periodontal tissues of a twelve-year-old girl, according to the guideline of the Institutional Board of Meikai University Ethics Committee (No. A0808)], after obtaining informed consent from the patients [17 (link)]. These cells were incubated for 48 h with the indicated concentrations of test samples or reference compounds sodium dichloroacetate (purchased from Sigma-Aldrich Chemical Company, Saint Louis, MO, USA), 5-fluorouracil (5-FU) from Kyowa (Tokyo, Japan), and doxorubicin (DXR) (St. Louis, MO, USA) and vehicle (DMSO) (0.008, 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, and 1%) in Dulbecco’s Modified Eagle Medium (DMEM) media, which was supplemented with 10% heat-inactivated fetal bovine serum [18 (link)]. Cell viability was determined by the MTT method [18 (link)]. Cytotoxicity caused by the vehicle (DMSO) was subtracted.
5
Culturing Human Cell Lines
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Human TSCC cell lines, HSC3 and SAS, were obtained from the Riken Cell Bank and the Cell Resources Center for Research (Tohoku University, Sendai, Japan), respectively. The human embryonic kidney cell line, HEK293T, and human fibroblast cell line, MRC5, were from the Riken Cell Bank and the Japanese Cancer Research Resources Bank, respectively, and cultured in DMEM containing 10% FBS (Sigma) at 7°C in a fully humidified atmosphere of 5% CO2 in air.
6
Establishing Cell Lines from Premolar Tooth
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Following informed consent from the patient at Meikai University Hospital, the 12-year-old girl had her first premolar tooth pulled from her lower jaw following Intramural Ethic Committee guidelines (No. A0808). The lower jaw first premolar tooth was used for establishing the HGF, HPLF, and HPC cells, and HOSCC cell lines [Ca9-22 (Catalog number: RCB-1976), HSC-2 (RCB-1945), HSC-3 (RCB-1975), and HSC-4 (RCB-1902)], purchased from Riken Cell Bank, Tsukuba, Japan. The Dulbecco’s modified Eagle’s medium (Catalog #: 30-2002), kanamycin (CAS #: 133-92-6), dimethyl sulfoxide (DMSO) (CAS #:67-68-5), and 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (CAS #: 298-93-1), and doxorubicin (CAS #: 25316-40-9) were purchased from FUJIFILM Wako Chem, Osaka, Japan. 5-fluorouracil (5-FU) (CAS #: 51-21-8) was purchased from Kyowa, Tokyo, Japan. Fetal bovine serum (FBS) (Catalog #: 16000044) and melphalan (CAS #: 148-82-3) were obtained from Sigma-Aldrich (St. Louis, MO, United States). The 100 mm dishes and 96-well plates used for the culture were purchased from True Line (Haryana, India) and TPP (Techno Plastic Products AG, Trasadingen, Switzerland), respectively.
7
Establishment of Sulfasalazine-resistant Cell Lines
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OSC19 cells were obtained from Kanazawa University as described previously [16 (link)]; HSC-2, HSC-3, and HSC-4 cells were from RIKEN Cell Bank; SCC25 cells were from DS Pharma Biomedical; and DMS114, HCT116 and 4T1 cells were from American Type Culture Collection. All cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and were maintained under 5% CO2 at 37° C. All cell lines were used within 6 to 12 months after receipt and were characterized by STR (short tandem repeat) analysis before use. For establishment of sulfasalazine-resistant OSC19 (OSC19-SSZR) cells, OSC19 cells were exposed to sulfasalazine at increasing doses up to 800 µM over 2 months. Cell resistance to xCT inhibitors was evaluated by measurement of cell viability.
8
Generating Cisplatin-Resistant HNSCC Cells
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The HNSCC cell line HSC-3 was obtained from Riken Cell Bank (Ibaraki, Japan). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Japan Ltd., Yokohama, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies Japan Ltd.) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin 25 µg/ml) at 37˚C in a humidified atmosphere containing 5% CO2.
To generate cisplatin-resistant cells, HSC-3 cells were initially cultured in medium containing 1 µM cisplatin; the cisplatin concentration was then slowly increased up to 100 µM. The generated cell line was viable in medium containing cisplatin at 200 µM for over 2 days.
To generate cisplatin-resistant cells, HSC-3 cells were initially cultured in medium containing 1 µM cisplatin; the cisplatin concentration was then slowly increased up to 100 µM. The generated cell line was viable in medium containing cisplatin at 200 µM for over 2 days.
9
Cytotoxicity Assay of Oral Cell Lines
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Cell culture. Human normal oral mesenchymal cells (HGF, HPLF, HPC) (13) at 10-18 population doublings were used in this study. Human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, derived from gingival tissue; HSC-2, HSC-3 and HSC-4 derived from tongue) were purchased from Riken Cell Bank (Tsukuba, Japan). All cells were cultured at 37˚C in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO 2 atmosphere. Cell morphology was examined periodically under a light microscope (EVOS FL; Thermo Fisher Scientific, Waltham, MA, USA).
Assay for cytotoxic activity. Cells were plated at 2×10 3 cells/0.1 ml in a 96-microwell plate. After 48 h, the medium was replaced with 0.1 ml of fresh medium containing different concentrations of single test compounds. Cells were incubated for 48 h and the relative viable cell number was then determined in triplicate by the MTT method, as described previously (7) (link)(8) (link)(9) (link)(10) (link)(11) (link). Control cells were treated with the same percentage of DMSO and the cell death induced by DMSO was subtracted. The concentration of compound that reduced the viable cell number by 50% (CC 50 ) was determined from the dose-response curve.
Assay for cytotoxic activity. Cells were plated at 2×10 3 cells/0.1 ml in a 96-microwell plate. After 48 h, the medium was replaced with 0.1 ml of fresh medium containing different concentrations of single test compounds. Cells were incubated for 48 h and the relative viable cell number was then determined in triplicate by the MTT method, as described previously (7) (link)(8) (link)(9) (link)(10) (link)(11) (link). Control cells were treated with the same percentage of DMSO and the cell death induced by DMSO was subtracted. The concentration of compound that reduced the viable cell number by 50% (CC 50 ) was determined from the dose-response curve.
10
Culturing HNSCC and HaCaT Cells
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Reagents and cell culture. The HNSCC cell lines HSC-2, HSC-3, SAS and Ca9-22 were obtained from the Riken Cell Bank (Ibaraki, Japan). The human immortalized non-tumorigenic keratinocyte cell line, HaCaT was purchased from DkFZ (Heidelberg, Germany). Cells were cultured in Dulbecco's modified Eagle's medium (DmEm) supplemented with 10% fetal bovine serum (FbS) (both from Life Technologies Japan Ltd., yokohama, Japan), 100 U/ml penicillin and 100 µg/ml streptomycin at 37̊C in a humidified atmosphere containing 5%.
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