HEK293T and HeLa cells were obtained from ATCC (CRL-1573 and CCL2, respectively). HEK293T and HeLa cells were cultured in DMEM (4.5 g/L glucose; ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Hyclone, GE), 6 mM L-glutamine, 1 mM sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin (ThermoFisher). All cells were grown at 37 °C in a humidified incubator maintained at 5% CO2 and tested bimonthly for mycoplasma using a MycoAlert detection kit (Lonza).
Dmem 4.5g l glucose
Manufactured by Thermo Fisher Scientific
Sourced in United States
DMEM (4.5g/L glucose) is a cell culture media formulation used to support the growth and maintenance of a variety of cell types in laboratory settings. It contains 4.5 grams per liter of glucose as a primary energy source for cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Mycoalert detection kit, by Lonza (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Transwell cell culture plate, by Corning (1 mentions)
Cos 1, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Caco 2 cells, by Leibniz Institute DSMZ (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Dmem f12 serum free medium, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
26 protocols using dmem 4.5g l glucose
1
Cell Culture Protocol for HEK293T and HeLa
2
Coculture Model for Glioblastoma Angiogenesis
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HUVECs (pooled donors) were purchased from ThermoFisher Scientific (Waltham, MA, USA), and grown in complete endothelial cell growth medium (EndoPan 3 Kit, Pan Biotech, Aidenbach, Germany). For the presented experiments, we used HUVECs between passage two and six. U87MG and A172 cells were purchased from ATCC and cultured in DMEM 4.5 g/L glucose (ThermoFisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (ThermoFisher Scientific).
Cells were grown at 37 °C in a humidified atmosphere of 5% CO2–95% air. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Cocultures of HUVEC and U87MG or A172 cells were performed using transwell cell culture plates (Corning, Corning, USA) with a polycarbonate membrane insert (0.4 µm pore size). Briefly, HUVECs and GBM cells were plated in the well and in the insert, respectively, in EndoPan 3/DMEM at 1:1 ratio. Forty-eight hours post-plating, cells were treated with a pulse of 25 µM Axitinib for 1 h. After extensive washings, GBM-cell-containing inserts were removed, and HUVECs were cultured in 100% complete EndoPan 3 medium for up to four days.
All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Axitinib and doxorubicin were resuspended in DMSO.
Cells were grown at 37 °C in a humidified atmosphere of 5% CO2–95% air. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Cocultures of HUVEC and U87MG or A172 cells were performed using transwell cell culture plates (Corning, Corning, USA) with a polycarbonate membrane insert (0.4 µm pore size). Briefly, HUVECs and GBM cells were plated in the well and in the insert, respectively, in EndoPan 3/DMEM at 1:1 ratio. Forty-eight hours post-plating, cells were treated with a pulse of 25 µM Axitinib for 1 h. After extensive washings, GBM-cell-containing inserts were removed, and HUVECs were cultured in 100% complete EndoPan 3 medium for up to four days.
All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Axitinib and doxorubicin were resuspended in DMSO.
3
Culturing COS-1 and Caco-2 Cells
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COS-1 (American Type Culture Collection, Mannasas, VA, United States), and Caco-2 cells (DSMZ, Braunschweig, Germany) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) 1 g/L glucose and DMEM 4.5 g/L glucose, respectively (ThermoFisher Scientific, Waltham, MA, United States), supplemented with 10% fetal calf serum (FCS) and penicillin–streptomycin (Sigma-Aldrich, Darmstadt, Germany). Both cell lines were passaged and maintained in a 5% CO2 humidified incubator at 37°C.
4
Collagen Hydrogel Fabrication and Characterization
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Collagen hydrogels,
with a final collagen concentration of 2.5, 4, and 6 mg/mL, were prepared
by diluting in an ice bath collagen type I solution (Rat Tail, stock
10.8 mg/mL, Corning, NY, US) in Dulbecco’s Modified Eagle Medium
(DMEM, 4.5 g/L glucose, Thermo Fisher Scientific, MA, US), 10×
Dulbecco’s phosphate buffered saline (DPBS), and 0.5 M NaOH
(both from Sigma-Aldrich, Germany) to adjust the pH to 7.4–7.6.
Supplemented hydrogels were fabricated diluting the collagen in 0.1%
LapNC-DMEM (working solution).
For characterization assays,
50 μL drops of the hydrogels were laid in wells (96 well plate).
To conduct permeability and spheroid growth tracking, the hydrogels
were gently pipetted inside the central culture chamber of a microfluidic
device (seesubsection 2.5.4 , and Figure 1 ). After placement in the respective container, collagen solutions
gelled for 20 min in a humid chamber at 37 °C. The temperature
and the pH conditions mentioned above induced a self-assembled gelation
process of the collagen hydrogels, where collagen fibers are physically
cross-linked.22 (link) During the gelation process,
collagen fibers create an interpenetrating polymer network embedding
the resuspended cells.23 (link)
with a final collagen concentration of 2.5, 4, and 6 mg/mL, were prepared
by diluting in an ice bath collagen type I solution (Rat Tail, stock
10.8 mg/mL, Corning, NY, US) in Dulbecco’s Modified Eagle Medium
(DMEM, 4.5 g/L glucose, Thermo Fisher Scientific, MA, US), 10×
Dulbecco’s phosphate buffered saline (DPBS), and 0.5 M NaOH
(both from Sigma-Aldrich, Germany) to adjust the pH to 7.4–7.6.
Supplemented hydrogels were fabricated diluting the collagen in 0.1%
LapNC-DMEM (working solution).
For characterization assays,
50 μL drops of the hydrogels were laid in wells (96 well plate).
To conduct permeability and spheroid growth tracking, the hydrogels
were gently pipetted inside the central culture chamber of a microfluidic
device (see
gelled for 20 min in a humid chamber at 37 °C. The temperature
and the pH conditions mentioned above induced a self-assembled gelation
process of the collagen hydrogels, where collagen fibers are physically
cross-linked.22 (link) During the gelation process,
collagen fibers create an interpenetrating polymer network embedding
the resuspended cells.23 (link)
5
Glioblastoma Cell Culture and Drug Treatment
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U87 (alias U87-MG) and A172 were purchased from ATCC and cultured in DMEM 4.5 g/L glucose (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). GSCs were isolated from patients’ surgical samples at the Institute of Neurosurgery, Catholic University of Rome as previously described [16 (link)] and grown in suspension in DMEM/F12 serum-free medium (Thermo Fisher Scientific) containing 2 mM L-glutamine, 0.6% glucose, 9.6 mg/mL putrescine, 6.3 ng/mL progesterone, 5.2 ng/mL sodium selenite, 0.025 mg/mL insulin, 0.1 mg/mL transferrin sodium salt (Sigma Aldrich, St Louis, MO, USA), EGF (20 ng/mL; Peprotech, London, UK), bFGF (10 ng/mL; Peprotech), and heparin (2 μg/mL; Sigma Aldrich) at 37 °C, 5% CO2. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Regorafenib (Bay73-4506) was purchased from Selleckem (Houston, TX, USA) and resuspended in DMSO.
Regorafenib (Bay73-4506) was purchased from Selleckem (Houston, TX, USA) and resuspended in DMSO.
6
Human GBM and Patient-Derived GSC Cell Lines
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The U87MG human GBM cell line was purchased from the American Type Culture Collection (Manassas, VA) and cultured in DMEM 4.5 g/L glucose (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (ThermoFisher Scientific). The patient-derived GSC1 and GSC275 cell lines were cultured under serum-free conditions [6 (link),7 (link)]. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2–95% air. Cells were regularly controlled to exclude mycoplasma contamination (Mycoalert Detection Kit, Lonza, Basel, Switzerland). Lentiviral transduction of green fluorescent protein (GFP) was performed as described [32 (link)].
7
Culturing HeLa and HL-60 Cell Lines
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Human cervical adenocarcinoma cells HeLa-Kyoto were cultured in DMEM, 4.5g/L glucose (61965-026,Thermofischer) supplemented with 10% Fetal Bovine Serum FBS (10270-106, Thermofischer) and 1% Penicillin Streptomycin (P/S, Life Technologies, Carlsbad, CA, USA) in a 5% CO2 incubator (Thermo Scientific, Waltham, MA, USA). HeLa Kyoto EGFP-LaminB1/H2B-mCherry cells from cell lines service (CLS, 330919) were used to image the nuclear membrane. Selection pressure for the stably expressed constructs was kept by adding 0.5mg/mL of G418 and 0.5ug/mL of puromycin in otherwise identical medium as described above. HL-60/S4 cells (ATCC Cat CRL-3306) were cultured in RPMI medium (ATCC-modification) supplemented with 10% FBS and 1% P/S in a 5% CO2 incubator. For all cell lines, number of passages was kept under 20. Our cells were authenticated by Microsynth and are mycoplasma-negative, as tested by GATC Biotech and are not on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee.
8
Culturing and Characterizing Human and Mouse Cell Lines
9
Cell Culture Protocols for Diverse Cell Lines
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HMEC-1, HMEC-GFP, NIH-3T3, and 3T3-GFP were maintained in DMEM 1g/L glucose (Invitrogen) supplemented with 10% foetal calf serum (FCS). Human dermal microvascular endothelial cells (HMVEC-d) and human umbilical vein endothelial cells (HUVEC) (Lonza) were maintained in endothelial growth medium (EGM-2-MV, Cambrex) supplemented with 5% FCS and additives recommended by the manufacturer. LLC/2 tumor cells were grown in DMEM 4.5g/L glucose (Invitrogen) supplemented with 10% foetal calf serum (FCS). CJ7 embryonic stem cells were cultured on 1% gelatin-coated dishes in Iscove's medium containing Glutamax (Iscove's modified Dulbecco's medium; Invitrogen) and supplemented with 15% FCS, 1% non-essential amino-acids, 1% ATAM, 150 μM monothioglycerol and 1000 U/mL of leukemia inhibitory factor (Chemicon). MCF-10A cells stably overexpressing oncogenic RasG12V were kindly provided to us by Dr O. Filhol (BCI lab, Grenoble, France). CCL-39 fibroblasts expressing a constitutively active Raf in an estrogen-responsive manner [29 (link)] were a generous gift from Dr G. Pagès (IRCAN, Nice, France). Unless otherwise indicated, antibodies were purchased from Cell Signaling, except for anti-phospho-MAPK (Promega), anti-MAPK (Sigma) and anti-c-Raf (BD Biosciences).
10
Cytotoxicity Assay with HeLa and RPE-1 Cells
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Human HeLa cells, a cell line derived from cervical adenocarcima, and RPE-1 cells, which are human retinal pigment epithelial cells, were obtained from the American Type Culture Collection (ATCC, Gainthersburg, MD, USA). HeLa cells were grown in RPMI 1640 medium with Glutamax (Gibco Invitrogen, Carlsbad, CA, USA) and RPE-1 cells were grown in DMEM, 4.5 g/L glucose (Gibco Invitrogen, Carlsbad, CA, USA). Both media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were maintained in a humid incubator at 37 °C in 5% CO2.
All chemicals, except those for which it is specified, were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).
Nocodazole, combretastatin-A4, colchicine and vinblastine were prepared at a 10 mM stock solution in Dimethyl sulfoxide (DMSO, #4540) aliquoted and stored at −20 °C.
All chemicals, except those for which it is specified, were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).
Nocodazole, combretastatin-A4, colchicine and vinblastine were prepared at a 10 mM stock solution in Dimethyl sulfoxide (DMSO, #4540) aliquoted and stored at −20 °C.
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