Human breast cancer cells (MDA-MB-231, BT549, SKBR3, Hs578T, BT-474, MDA-MB-468, SUM-159, MDA-MB-453 and MCF-7) were obtained from American Type Culture Collection (ATCC). Those cells were routinely cultured in DMEM or RPMI 1640 (Gibco-BRL, Australia) comprising 10% fetal bovine serum (FBS; Gibco BRL, Australia), 100 U/ml penicillin and 100 mg/ml streptomycin. For hypoxic cell culture, cells were cultured at 37 °C in a humidified tri-gas incubator containing 5% CO2 and 1% O2. Controls were cultured at 37 °C in a standard humidified incubator containing 5% CO2 and 20% O2. For creatine pretreatment, cells were cultured in media containing 1% FBS and 5 mM creatine (Sigma-Aldrich, USA) 24 h ahead of experiments.
Creatine
Manufactured by Merck Group
Sourced in United States, Germany
Creatine is a naturally occurring organic acid that plays a key role in energy production within cells. It is a compound found in muscle tissue and is involved in the recycling of adenosine triphosphate (ATP), the primary energy currency of cells. Creatine is an important component of the phosphocreatine system, which helps maintain ATP levels during periods of high energy demand.
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Lab products found in correlation
Creatinine, by Merck Group (10 mentions)
Taurine, by Merck Group (9 mentions)
Carnitine, by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
L carnitine, by Merck Group (6 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
Pyruvate, by Merck Group (4 mentions)
Cholesterol, by Merck Group (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
B27 with vitamin a, by Thermo Fisher Scientific (3 mentions)
Normocin, by InvivoGen (3 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (3 mentions)
4 nitrobenzoic acid, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Uridine, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Gentamycin, by Thermo Fisher Scientific (3 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (2 mentions)
Cortisol, by Merck Group (2 mentions)
55 protocols using creatine
1
Hypoxia and Creatine Effects on Breast Cancer Cells
2
Multi-pH CEST Gel Phantom for Creatine Measurement
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A multi-pH CEST gel phantom composed of creatine and low gelling point (LGP) agarose (Sigma-Aldrich, St. Louis, MO, USA) was prepared, as previously described (20 (link)). In brief, 1% agarose was added to phosphate-buffered saline (PBS; Oxoid, Basingstoke, UK). The phantom was microwave-heated to boiling and then cooled for ~25 mins in a water bath set at 50°C (Cole-Palmer, Vernon Hills, IL, USA). creatine (Sigma-Aldrich) was then added to the gel solution to final concentrations of 50, 100 and 200 mM, followed by serial titration to pH 7.0 (EuTech Instruments, Ayer Raja, Singapore). The solutions were transferred into separate 10 ml centrifuge tubes for each creatinine concentration, which were then sealed and inserted into a phantom holder (63 mm Swift Cradle; Agilent Technologies, Inc., Santa Clara, CA, USA). A central tube filled with 1% agarose-gel in the absence of creatine was used as a background control.
3
NMR Metabolomics Substrate Preparation
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DSS [3-(Trimethylsilyl)-1-propanesulfonic acid] sodium salt, L-aspartic acid,
L-glutamic acid, creatine, deuterium oxide, deuterium oxide including TSP
[3-(trimethylsilyl)propionic-2,2,3,3-d4 acid]
sodium salt, HEPES [4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid], and
mono- and di-phosphate sodium salt (anhydrous form) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). MOPS (3-Morpholinopropanesulfonic acid) was
purchased from Amresco (Solon, OH, USA).
L-glutamic acid, creatine, deuterium oxide, deuterium oxide including TSP
[3-(trimethylsilyl)propionic-2,2,3,3-d4 acid]
sodium salt, HEPES [4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid], and
mono- and di-phosphate sodium salt (anhydrous form) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). MOPS (3-Morpholinopropanesulfonic acid) was
purchased from Amresco (Solon, OH, USA).
4
Metabolite Quantification by LC-MS
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All water was purified using a Milli-Q™ water system from MilliPore (Bedford, MA, USA). Ammonium formate (LCMS-grade) and formic acid (LCMS-grade) were obtained from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (LCMS-grade) was purchased from Fisher Scientific (Zurich, Switzerland). Acetonitrile (ACN) used for protein precipitation was kept on ice throughout the sample preparation procedure. Butyryl-l -carnitine, creatine, O-acetyl-l -carnitine hydrochloride, and stachydrine-(dimethyl-13C2) monohydrate were obtained from Sigma-Aldrich.
5
Mitochondrial Respiration Measurement
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Oxygen consumption was measured polarographically in water-jacketed respiration chambers maintained at 37 °C (Oxygraph, Hanstech Instruments, King’s Lynn, UK) as previously detailed [56 (link)]. Following daily calibration with Buffer Z [50 mM K-MES (Sigma M0895), 30 mM KCl (Sigma P4504), 10 mM K2HPO4 (Fisher, Hampton, NH, USA, P290), 1 mM EGTA (Sigma E4378), 5 mM MgCl2-6H2O (Sigma M2670), 0.005 mM Glutamate (Sigma G8415), 0.002 mM Malate (Sigma M6773), and 0.05% BSA (Sigma A6003), pH to 7.1 at 4 °C] and Na2SO4, 10 µL of isolated SS or IMF mitochondria was independently resuspended in 965 µL of buffer Z, containing 20 mM creatine (Sigma C0708) warmed to 37 °C within the oxygraph chamber. Mitochondria were allowed to equilibrate before the addition of 10 µL Malate (272.21 mM; Sigma M7397) and 10 µL pyruvate (500 mM; Sigma P5280) followed by the addition of 5 µL of ADP (48.09 mM; MP Biomedicals, Irvine, CA, USA, 150259) to determine state 3 and state 4 respiration. Respiratory control ratio (RCR) was designated as state 3 respiration divided by state 4 respiration. Values were normalized post hoc to protein content by the Bradford method.
6
Creatine Supplementation in Pregnant Rats
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Pregnant dams were fed either a control diet of standard rat and mouse chow (2.16mg creatine/g) throughout pregnancy, or chow supplemented with 5% creatine monohydrate (32.44 mg creatine/g; Specialty Feeds, Glen Forrest, Perth, Australia; creatine, Sigma) from day 20 of gestation to term (day 39). Water was freely available and animals were housed in family groups.
7
Optimized neuronal culture media
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BrainPhys neuronal media (Stem Cell Technologies) supplemented with 1X B27 with vitamin A (ThermoFisher), 1X N2 Plus media supplement (R&D Systems), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mM creatine (Sigma-Aldrich), 200 nM L-ascorbic acid (Sigma-Aldrich), 1 μg/ml mouse laminin (ThermoFisher), 0.5 mM glutamax (ThermoFisher), 0.5X penicillin-streptomycin (ThermoFisher), 1X Normocin (Invivogen), 5 ng/ml TGF-b (Peprotech), 100 ng/ml human IL-34 (Peprotech), 1.5 μg/ml cholesterol (Sigma-Aldrich), 1 ng/ml gondoic acid (Cayman Chemicals), 100 ng/ml oleic acid (Cayman Chemicals), 460 μM Thioglycerol (Sigma-Aldrich), 1X Insulin-Transferrin-Selenium (ThermoFisher), 25 ng/ml rhM-CSF (Peprotech), and 5.4 μg/ml Human Insulin Solution (Sigma).
8
Creatine-Enriched CHO Mixture
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Two percent creatine(Sigma-Aldrich USA) was mixed into the normal CHO using an electric mill. After adding some water and cutting the paste, the creatine and CHO mixture were dried in a 30-45 minute exposure to a continuous warm air stream. After injection, animals in the AdCr+ group had free access to this creatine-CHO mixture for six weeks.
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Two percent creatine(Sigma-Aldrich USA) was mixed into the normal CHO using an electric mill. After adding some water and cutting the paste, the creatine and CHO mixture were dried in a 30-45 minute exposure to a continuous warm air stream. After injection, animals in the AdCr+ group had free access to this creatine-CHO mixture for six weeks.
9
Hypoxia Response to Creatine Compounds
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Lvm3b cells were seeded at 300,000 cells per well in a six-well plate. After 24-hour incubation under normoxia, the cells were cultured under hypoxia (0.5% oxygen, 5% CO2, 37°C) for 96 hours in the presence of RGX-202, creatine (Sigma-Aldrich, #C3630), or phosphocreatine (Sigma-Aldrich, #237911) at 10 μM. The medium was replaced, and incubation continued up to 120 hours when cells were counted.
10
LC-MS Quantification of Metabolites
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