The rat pheochromocytoma cell line, PC12, was obtained from the Cell Resource Center of Yangzhou University, China. The cryopreserved PC12 cells were resuscitated and seeded in culture dishes with Dulbecco’s modified Eagle’s medium (Gibco, Suzhou, China), containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) (Xiao et al., 2012). The cells were cultured in an incubator with 5% CO2 at 37°C and the medium was replaced every 2 days. Cells were treated with 0.25% trypsin (Gibco, Grand Island, NY, USA) and seeded into culture dishes at 7.5 × 104 cells/cm2 for subsequent experiments. According to previous methods (Guo et al., 2013), chemical hypoxia was induced by adding cobalt chloride (CoCl2; Sigma, St. Louis, MO, USA) at 250 μM to the medium. Cells were treated with CoCl2 for different times (1, 3, 6, 12 and 24 hours). The PC12 cells cultured in normal condition were assigned to control group. Cell proteins were collected for analysis.
Cobalt chloride cocl2
Manufactured by Merck Group
Sourced in United States, Germany, Macao, Italy
Cobalt chloride (CoCl2) is an inorganic compound that is commonly used as a laboratory reagent. It is a crystalline solid that is soluble in water and various organic solvents. Cobalt chloride is known for its ability to change color in response to changes in humidity, making it useful as an indicator of moisture content.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Mg132, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Poly d lysine, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Hbm msc, by Lonza (2 mentions)
Desferrioxamine dfx, by Merck Group (2 mentions)
Hct116, by American Type Culture Collection (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
75 protocols using cobalt chloride cocl2
1
Cobalt Chloride-Induced Hypoxia in PC12 Cells
2
Hypoxic Pancreatic Cancer Cell Culture
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The human pancreatic cancer cell lines Aspc‐1, Bxpc‐3, CFPAC‐1, MiaPaCa‐2, PANC‐1 and SW1900 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). MiaPaCa‐2, PANC‐1 were maintained in DMEM supplemented with 10% foetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified incubator containing 5% CO2 in air at 37°C. Aspc‐1, Bxpc‐3, CFPAC‐1 and SW1900 were maintained in RPMI 1640 containing supplements as above. For all experiments, the cell lines were treated in serum‐free medium (0.1% bovine serum albumin, 5 mg/ml transferrin and 5 ng/ml sodium selenite and antibiotics). Hypoxic conditions (1% O2) were created with a three‐gas incubator MiniGalaxy A (RS Biotech, Irvine, UK) by injection of N2 (1% O2/94% N2/5% CO2 atmosphere, 37°C). In other experiments hypoxia mimetic conditions were chemically generated by treating cells with 100 mM cobalt chloride (CoCl2; Sigma‐Aldrich, St. Louis, MO, USA) for the indicated times.
3
Culturing Human Cervical Carcinoma and 293T Cells
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Human cervical carcinoma cell line SiHa, and 293T/17 cells were purchased from the American Type Culture Collection (ATCC), and cultured in DMEM (Hyclone, Thermo scientific, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) and 1% penicillin-streptomycin solution (Hyclone, Thermo scientific, USA) at 37ºC in a 5% CO2 incubator. For hypoxia treatment, cells were incubated in a humidified O2 control incubator (5% CO2, 1% O2) for indicated time or cultured with cobalt chloride (CoCl2, Sigma) solution as reported previously 16 (link).
4
Purification and Hypoxic Stress Assay of Retinal Ganglion Cells
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Primary cultured RGCs were purified by two-step immunopanning method as we described previously (Gao et al., 2016 (link)). RGCs were collected and seeded into 96, 24 and 6-well plates pretreated with mouse-laminin (Trevigen Inc., Gaithersburg, MD, USA) and poly-D -lysine (Sigma–Aldrich, St. Louis, MO, USA). Plates were incubated in a humidified incubator with 5% CO2 at 37° C. RGCs purity was checked by Thy1.1 and Brn 3b immunocytochemical staining (about 85%) (Gao et al., 2016 (link)). Forty-eight hours after seeding, RGCs were incubated with 200 μM cobalt chloride (CoCl2, Sigma–Aldrich, St. Louis, MO, USA) to induce hypoxia and apoptosis (Kim et al., 2013 (link)), combined with 0, 10, 20, 50, or 100 ng/ml MANF for 24 h, then incubated 12 h, 24 and 48 h under optimal concentration. Half of the RGC medium was replaced with fresh RGC medium every 3 days.
5
Triptolide and Hypoxia Signaling Modulation
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MIA PaCa-2 cells (ATCC) were cultured in DMEM medium (Hyclone) supplemented with 10% FBS and 1% penicillin streptomycin. S2-VP10 cells were cultured in RPMI 1640 medium (Hyclone) with 10% FBS and 1% penicillin streptomycin. Triptolide was used at a concentration of 50 nM in all in vitro experiments based on the response of this compound from our previous studies18 (link), 20 (link), 21 (link) (unless otherwise mentioned). The hypoxia mimetic cobalt chloride (CoCl2) (Sigma) was used at a concentration of 200 uM for HIF-1α reporter assays. The proteasome inhibitor MG-132 (Sigma) was used at a concentration of 10 uM for HIF-1α reporter assays. BAY87-2243, a hypoxia inhibitor was used in indicated concentration. Minnelide, the pro drug of triptolide, was used in vivo at indicated concentrations.
6
Synthesis of Lithium-Based Compounds
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Cobalt chloride (CoCl2) (dry or hydrated with two H2O), lithium phenoxide (LiOPh) in tetrahydrofuran (THF), lithium iso-propoxide (LiOiPr) in THF, ethanol (technical grade and analytical grade), tetramethylethylenediamine ( TMEDA), dioxane, dimethoxyethane (DME), pyridine (Py), heptane and micron-sized HT-LiCoO2 were purchased from Sigma-Aldrich (Switzerland). Lithium tert-butoxide (LiOtBu) in THF, lithium methoxide (LiOMe) in methanol, lithium ethoxide (LiOEt) in THF and THF (dry and over molecular sieves) were purchased from Acros Organics (Belgium). Deionized water was produced in house by double distillation.
7
Culturing Melanoma and HEK293T Cells
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The human melanoma cell line and A2058, SK-MEL-2, and SK-MEL-28 were purchased from the American Type Culture Collection (ATCC). The melanoma cells were cultured in Gibco Minimum Essential Media (WelGene, Daegu, Republic of Korea). HEK293T cells were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA). Both media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell lines were maintained in 5% CO2 at 37 °C in humidified incubators and subcultured every 3–4 days. Hypoxia was induced using cobalt chloride (CoCl2) (Sigma-Aldrich, St. Louis, MO, USA) or by placing the cells in a hypoxia induction chamber (#27310, STEMCELL Technologies Inc., Vancouver, BC, Canada) loaded with mixed gas containing 1% O2, 5% CO2, and 94% N2.
8
Serum Albumin-Bound Cobalt Assay
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The procedure was based on prior description by Bar-Or et al. [18 (link)]. In short, 100 μL of serum was mixed with 25 μL cobalt chloride (CoCl2, 1 mg/mL) (Sigma-Aldrich, USA) in a 96-well plate and incubated at room temperature for 10 minutes. 25 μL dithiothreitol (DTT, 1.5 mg/mL) (Sigma-Aldrich, USA) was added, followed by 2-minute incubation to allow the reaction with free cobalt salt. Finally 150 μL saline was added to terminate the reaction. Absorbance was measured by a spectrophotometer Synergy H1 (BioTek, USA) at 470 nm. The IMA was expressed as absorbance unit, which equaled the absorbance of the testing well minus the absorbance of control well with no DTT added. High absorbance value indicated more free cobalt salt reacting with DTT; therefore fewer cobalt salt was bound to albumin.
9
Cobalt Chloride Induces Hypoxia in HIBEpiCs
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The human intrahepatic bile duct epithelial cells (HIBEpiCs) were purchased from ICell Bioscience Inc. (Shanghai, China). The human hepatocellular carcinoma cell line MHCC-97h and human mononuclear cell line THP-1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The HIBEpiC and THP-1 were cultured in RPMI-1640 medium (Gibco, Grand Island, USA), and MHCC-97h was cultured in DMEM medium (Gibco, Grand Island, USA), all media containing 10% fetal bovine serum (Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). All three cell lines were grown in an incubator (37°C, 5% CO2).
Cobalt chloride (CoCl2, Sigma-Aldrich, USA) was used to simulate the hypoxia process of HIBEpiCs. The HIBEpiCs were divided into four groups: the control group (CON), the IL-22 (10 ng/ml) (Recombinant human IL-22, Absin, Shanghai, China) treatment group, the CoCl2 (150 μM) treatment group, and the CoCl2 (150 μM)+IL-22 (10 ng/ml) treatment group.
Cobalt chloride (CoCl2, Sigma-Aldrich, USA) was used to simulate the hypoxia process of HIBEpiCs. The HIBEpiCs were divided into four groups: the control group (CON), the IL-22 (10 ng/ml) (Recombinant human IL-22, Absin, Shanghai, China) treatment group, the CoCl2 (150 μM) treatment group, and the CoCl2 (150 μM)+IL-22 (10 ng/ml) treatment group.
10
Breast Cancer Drug Combination Effects
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The following drugs were used: letrozole (Novartis, NY, USA); lapatinib (GlaxoSmithKline Pharmaceutical, Brentford, Middlesex, United Kingdom); trastuzumab (Genentech, San Francisco, CA); exemestane (Pfizer, NY, USA); cycloheximide (#C1988), actinomycin D (#A9415), and cobalt chloride (CoCl2; #C8661) (all from Sigma, St. Louis, MO). The following antibodies were used in western blot analyses: HER2 (#04-1127) and BCRP (EMD Millipore, Billerica, MA); HIF-1α (#610959; BD Biosciences); ERα (#8644; Cell Signaling Technology, Danvers, MA); phosphorylated and total ERK1/2, Akt (#4058 and #4685), mTOR (#2971 and #2972) and p70 S6 kinase (#9205 and #9202) all from Cell signaling Technology, Danvers, MA); and β-actin (#4970; Cell Signaling Technology, Danvers, MA).
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