MSCs from human dental pulp were expanded as described previously [8 (link)] and transduced with lentiviral vectors pWP-green fluorescent protein (GFP-MSCs) or pWP-HIF-GFP (HIF-MSCs) as previously described [7 (link)]. Transduction efficiency was evaluated by flow cytometry (Coulter EPICS XL flow cytometer; Beckman Coulter) to determine the percentage of GFP-positive cells. The percentages of infection obtained were normally around 90%. Human dental pulp MSCs (n = 3; Inbiobank) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Sigma-Aldrich, Spain) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Life Technologies, Thermo Fisher Scientific). Cells were grown until confluence and subsequently subcultured up to 12 passages. For stimulation assays, 5 × 104 MSCs were seeded in six-well flat-bottom culture plates and, after 3 days, media were replenished and supplemented or not with 10 ng/ml recombinant human interferon (rhIFN)-gamma (Invitrogen). After 15 h, cells were lysed for subsequent analysis by quantitative polymerase chain reaction (qPCR).
Dulbecco s modified eagle s medium dmem low glucose
Manufactured by Merck Group
Sourced in United States
Dulbecco's modified Eagle's medium (DMEM) low glucose is a cell culture medium used to support the growth and maintenance of various cell lines. It is a basal medium that provides essential nutrients, amino acids, and other components required for cell proliferation. The low glucose concentration in this formulation is suitable for cell types that are sensitive to high glucose levels.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (2 mentions)
Heat inactivated fetal calf serum fcs, by Thermo Fisher Scientific (1 mentions)
Coulter epics xl flow cytometer, by Beckman Coulter (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
2 thiobarbituric acid, by Merck Group (1 mentions)
Dmem ham f12, by Merck Group (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Folin ciocalteu reagent, by Merck Group (1 mentions)
2 4 6 tripyridyl s triazine, by Merck Group (1 mentions)
Collagenase type 5, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Nape pld, by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Avantor (1 mentions)
12 protocols using dulbecco s modified eagle s medium dmem low glucose
1
Expansion and Transduction of Dental Pulp MSCs
2
Antioxidant and Cytotoxicity Assays
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All the solutions were prepared with ultrapure water (Millipore, São Paulo, Brazil). Folin–Ciocalteu reagent, 2-thiobarbituric acid, 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), iron chloride(III) hexahydrate, copper(II) sulfate pentahydrate, acetonitrile, high-performance liquid chromatography analytical standards (Gallic, syringic, 2-hydroxycinnamic, protocatechuic, 2,4-dihydroxybenzoic, 2,5-dihydroxybenzoic, p-coumaric, 5-O-caffeoylquinic, caffeic, ferulic, rosmarinic, and ellagic acids; quercetin-3-rutinoside, procyanidin A2, (−)-epicatechin, (+)-catechin, and quercetin), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Dulbecco’s modified Eagle’s medium (DMEM)/low glucose, DMEM/Ham-F12, DCFH-DA (dichlorofluorescein diacetate), penicillin, and streptomycin were purchased from Sigma-Aldrich (São Paulo, Brazil). 2,4-Dihydroxybenzoic and 2,5-dihydroxybenzoic acids were purchased from Carl Roth (Karlsruhe, Germany). Ethyl alcohol 99.8%, methyl alcohol 99.8%, petroleum ether, ascorbic acid, and glacial acetic acid were purchased from Pró-Análise (Porto Alegre, Brazil). A549, HCT8, and IMR90 (healthy lung fibroblast cells) cell lines were obtained from the Rio de Janeiro cell bank (BCRJ, Rio de Janeiro, Brazil).
3
Synthetic Cannabinoid Receptor Assay
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BAR-1 was synthetized by Navarrete-Vázquez and colleagues20 , and its synthesis has been described previously. Dulbecco’s modified Eagle’s medium (DMEM) low glucose, Fetal bovine serum (FBS), penicillin/streptomycin, l -glutamine, collagenase type V, Histopaque 1077, STZ and PCR primers for CB1r, CB2r, MAGL, NAPE-PLD, FAAH, DAGL and 18s rRNA were purchased from Sigma Aldrich (St. Louis MO, USA). Preproinsulin (PPI) and preproglucagon (PPG) were obtained from Qiagen (West Sussex, UK). Real-time PCR master mix and reagents were purchased from Fermentas/Thermo Scientific (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA). A1cNow kit was from ChekDiagnostics (Diagnodistributions, USA), and insulin and glucagon enzyme-linked immunosorbent assay (ELISA) kits were obtained from Alpco (Salem, NH, USA). Anti-insulin primary antibody and secondary antibodies were obtained from Santa Cruz Biotechnology.
4
Curcumin-Loaded PS-PAA Nanoparticles
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Polystyrene
(PS, 35 kDa), poly(styrene)–block– poly(acrylic acid) (PS-PAA) (Mw = 83
500, Mw of
PS block = 70 500, Mw of PAA block = 13
000, and PDI ≤1.1), curcumin (Cur, curcuminoid content ≥
94%, Cur content ≥ 80%), oleic acid (OA, 90%), Dulbecco’s
modified Eagle’s medium (DMEM)—low glucose, MTT suitable
for cell culture (≥97.5% HPLC), and PBS (tablets) were purchased
from Sigma-Aldrich and used as received. Poly(vinyl alcohol) (PVA, Mw 14 000, 98.5–100% degree of hydrolysis)
was obtained from BDH Chemicals. Sodium dodecyl sulfate (SDS, ≥
99%) was purchased from BioSchop. Millipore-quality water was used
in all experiments.
(PS, 35 kDa), poly(styrene)–block
500, Mw of
PS block = 70 500, Mw of PAA block = 13
000, and PDI ≤1.1), curcumin (Cur, curcuminoid content ≥
94%, Cur content ≥ 80%), oleic acid (OA, 90%), Dulbecco’s
modified Eagle’s medium (DMEM)—low glucose, MTT suitable
for cell culture (≥97.5% HPLC), and PBS (tablets) were purchased
from Sigma-Aldrich and used as received. Poly(vinyl alcohol) (PVA, Mw 14 000, 98.5–100% degree of hydrolysis)
was obtained from BDH Chemicals. Sodium dodecyl sulfate (SDS, ≥
99%) was purchased from BioSchop. Millipore-quality water was used
in all experiments.
5
Cytotoxicity Evaluation of Chemical Compounds
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TCS (triclosan; 5-chloro-2-(2,4-dichlorophenoxy) phenol; CAS 3380-34-5), DEHP (bis(2-Ethylhexyl) phthalate; CAS 117-81-7), 3,4-benzopyrene (B[a]P; CAS 50-32-8), methyl methanesulfonate (MMS; CAS 66-27-3), cytochalasin B (Cyt-B; CAS 14930-96-2), dimethyl sulfoxide (DMSO; CAS 67-68-5), Giemsa (CAS 51811-82-6) and Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose) were obtained from Sigma-Aldrich (St. Louis, MO, United States), Trypsin-EDTA (0.25%) and antibiotic-antimycotic solution (10,000 units/mL of Penicillin, 10,000 μg/mL of Streptomycin, and 25 μg/mL of Amphotericin B) came from Gibco (Grand Island, NE, United States) and fetal bovine serum (FBS) was purchased from LGC Biotecnologia (Cotia, Brazil). All other chemicals, reagents, and buffers were analytical grade products from Sigma-Aldrich (St. Louis, MO, United States).
6
Isolation of Normal Mononuclear Cells
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Normal mononuclear cells were offered by Dr. Marwan El-Sabban’s Lab at the American University of Beirut (AUB) as a kind gift. The normal MNCs were obtained originally from bone marrow aspirate leftovers of healthy patients attending AUB Medical center (AUB-MC). BM aspirates were centrifuged on Ficoll/Hypaque (GE Healthcare Life Sciences, Uppsala, Sweden), a density gradient step to separate MNCs from red blood cells and neutrophils. Then the buffy coat was aspirated and seeded in petri dishes using Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% FBS (Gibco, Dublin, Ireland) and 100 U/mL penicillin and 100 µg/mL streptomycin (Lonza, Basel, Switzerland) in a humidified incubator at 37 °C and 5% CO2. One week later, the cells in suspension were collected as a purified MNC population and cultured in the same conditions as detailed by Zibara et al. [25 (link)].
7
Primary Culture of Human Skin Cells
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Primary cultures of human dermal fibroblasts and keratinocytes were obtained from skin samples of patients undergoing abdominal surgery, as described in [56 (link)]; cells were cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and high-glucose DMEM (both from Sigma-Aldrich, St Louis, MO, USA), respectively, supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA, USA), along with 100 U mL−1 penicillin and 100 µg mL−1 streptomycin (P/S, Millipore, Bedford, MA, USA). Primary cultures of human umbilical cord vein endothelial cells (HUVEC, ATCC CRL-1730) were obtained from Lonza (Basel, Switzerland) and were grown in Endothelial Cell Growth Medium-2 (EGM-2) BulletKitTM (Lonza). All cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.
8
HPLC and Cell Culture Protocols
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HPLC grade solvents (dichloromethane, ethyl acetate and acetonitrile LiChrosolv®), Mass-Spectrometry grade (acetonitrile LiChromsolv® hypergrade) solvents and ammonium acetate were acquired from Merck (Germany) and used without further purification. The TLC silica gel 60 F254 plates from Merck (Germany) were employed to perform preliminary chromatographic fingerprinting.
Chemical and reagents used in cell culture were PBS 1X (Gibco, Carlsbad, CA, USA), low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640 Medium (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), penicillin–streptomycin (P/S) 10,000 U/Ml (Invitrogen, Carlsbad, CA, USA), glutamine (Gibco, Carlsbad, CA, USA), methyl thiazole tetrazolium (MTT, Amresco, Solon, OH, USA) assay.
Chemical and reagents used in cell culture were PBS 1X (Gibco, Carlsbad, CA, USA), low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640 Medium (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), penicillin–streptomycin (P/S) 10,000 U/Ml (Invitrogen, Carlsbad, CA, USA), glutamine (Gibco, Carlsbad, CA, USA), methyl thiazole tetrazolium (MTT, Amresco, Solon, OH, USA) assay.
9
Primary Hepatocyte Lipid Metabolism Assay
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The fluorescent dyes 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), propidium iodide (PI), Fura-2 AM, and JC-1 were purchased from Invitrogen (Carlsbad, CA, USA). The calcium-specific chelator BAPTA-AM was also obtained from Invitrogen. The fatty acids palmitate and oleate, bovine serum albumin (BSA), and low glucose Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma Aldrich (St. Louis, MO, USA). Primary hepatocytes were cultured on plates coated with Collagen I (Rat Tail) from BD Biosciences (San Jose, CA).
10
Osteogenic Differentiation of Murine BMSCs
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After euthanasia, femurs and tibiae of 6–9 week-old wild-type and Runx2+/− mice were carefully cleaned from adherent soft tissue and bone marrow cells were harvested. Collected cells were seeded at a density of 4 × 107 cells per 3.5 cm tissue culture dish (BD Falcon) and cultured in growth medium: Dulbecco's modified Eagle's medium-low glucose (DMEM; Sigma) containing 10% heat-inactivated fetal bovine serum (FBS; Nichirei) and penicillin/streptomycin (100 IU/ml and 100 μg/ml; Sigma), at 37 °C in 5% CO2 atmosphere. After 4 days of culture, nonadherent cells were removed and adherent cells were cultured 3 more days until 90% confluence to use as BMSCs in this study. For promoting osteogenesis, BMSCs were cultured in osteogenic medium: growth medium supplemented with 10 nM dexamethasone (Sigma), 82 μg/ml l -ascorbic acid (Wako) and 10 mM β-glycerophosphate (Sigma), at 37 °C in 5% CO2 atmosphere. Osteogenic medium was changed every three days.
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