HeLa and BSC-1 cells were grown at 37°C under 5% CO2 in DMEM high glucose Glutamax (Invitrogen) supplemented with 10% FCS, 0.01% penicillin-streptomycin, and 5 mM pyruvate. Genome-edited SK-MEL-2 expressing RFP-tagged clathin light chain were kindly provided by David Drubin (University of California, Berkeley), and were grown at 37°C under 5% CO2 in DMEM/F12 (Invitrogen) supplemented with 10% FCS, 0.01% penicillin-streptomycin, and 5 mM pyruvate. HeLaM cells stably expressing Mito-YFP-FRB were kindly provided by Margaret S. Robinson (Cambridge Institute for Medical Research). These were grown at 37°C under 5% CO2 in DMEM high glucose Glutamax (Invitrogen) supplemented with 10% FCS, 0.01% penicillin-streptomycin, 5 mM pyruvate, and 137.5 µg/ml hygomycine B. HeLaM Mito-YFP-FRB cells stably expressing C-terminally GFP-FKBP-tagged rat endoA2 were generated for this study (see below) and grown in the same medium than the mother cells, supplemented with 0.5 mg/ml G418.
Dmem high glucose glutamax
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, France
DMEM high glucose GlutaMAX™ is a cell culture medium formulated to support the growth and maintenance of various cell lines. It contains high glucose concentration and is supplemented with the GlutaMAX™ dipeptide, which serves as a stable glutamine substitute. This medium is designed to provide the essential nutrients and components required for cell proliferation and survival in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (19 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Gentamycin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
High dose tricaine, by Merck Group (4 mentions)
Matrigel, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lumar v12 stereomicroscope, by Zeiss (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (2 mentions)
L proline, by Merck Group (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
57 protocols using dmem high glucose glutamax
1
Cell Culture Conditions and Genome Editing
2
Isolation and Culture of Zebrafish Muscle Fibers
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Isolation and culture of zebrafish adult muscle fibres was previously described (Ganassi et al., 2018 (link)). Zebrafish aged 8–9 months (adult) or 1 month (juvenile) were culled in high-dose tricaine (Sigma Aldrich), immersed for 5 min in 1% Virkon (3S Healthcare) diluted in dH2O, washed in PBS for 5 min followed by 70% ethanol rinse, eviscerated and skinned. Fifteen-month-old adult were used for the myogfh265 allele. For myofibre dissociation, trunk muscle was incubated in 0.2% Collagenase (C0130, Sigma Aldrich), 1% Penicillin/Streptomycin DMEM supplemented with 50 µg/ml gentamycin (Thermo Fisher) at 28.5°C for at least 2 hours. Single muscle myofibres were released by trituration using heat-polished glass pipettes and washed three times with DMEM Glutamax High Glucose (Gibco). Myofibres were then imaged for total length measure or plated on Matrigel (Invitrogen) coated 96- or 24-well plates and cultured in growth medium (20% Fetal Bovine Serum in 1% Penicillin/Streptomycin/DMEM Glutamax High Glucose supplemented with 10 µg/ml gentamycin). At indicated time points, MPCs were washed twice with PBS to remove plated myofibres, EdU pulsed (10 μM, Invitrogen Life Technologies) for 8 hr in fresh media and then fixed with 4% PFA for 15 min. For immunostaining myofibres were fixed in 4% PFA in PBS immediately after dissociation to reduce processing time and avoid MuSC activation.
3
Isolation and Culture of Zebrafish Muscle Fibers
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Isolation and culture of zebrafish adult muscle fibres was previously described (Ganassi et al., 2018) . Zebrafish aged 8-9 months (adult) or 1 month (juvenile) were culled in high dose tricaine (Sigma Aldrich), immersed for 5 min in 1% Virkon (3S Healthcare) diluted in dH2O, washed in PBS for 5 min followed by 70% ethanol rinse, eviscerated and skinned. 15 month old adult were used for the myog fh265 allele. For myofibre dissociation, trunk muscle was incubated in 0.2% Collagenase (C0130, Sigma Aldrich), 1% Penicillin/Streptomycin DMEM supplemented with 50 µg/ml gentamycin (Thermo Fisher) at 28.5 °C for at least two hours. Single muscle myofibres were released by trituration using heat-polished glass pipettes and washed three times with DMEM Glutamax High Glucose (Gibco). Myofibres were then imaged for total length measure or plated on Matrigel (Invitrogen) coated 96 or 24 well plates and cultured in growth medium (20% Fetal Bovine Serum in 1% Penicillin/Streptomycin/ DMEM Glutamax High Glucose supplemented with 10 µg/ml gentamycin). At indicated time points, MPCs were washed twice with PBS to remove plated myofibres, EdU pulsed (10 μM, Invitrogen Life Technologies) for 8 hours in fresh media and then fixed with 4% PFA for 15 minutes. For immunostaining myofibres were fixed in 4% PFA in PBS immediately after dissociation to reduce processing time and avoid MuSC activation.
4
Oligodendrocyte Progenitor Cell Culture
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The OPCs obtained with both methods were seeded at a density of approximately 3 × 104 cells/cm2 into 24-well plates (Nunc, ThermoFisher) with poly-l -lysine-coated glass coverslips for immunofluorescencent staining or 6-well plates (Nunclon) for qPCR and cultured under physiological normoxia (37 °C, 5% O2, 5% CO2).
In order to evaluate the effect of different compositions of culture medium on OPCs differentiation, cells obtained with the MB method after 2 days in culture were then cultured in different media: ‘PROLIF – PDGFR-AA and FGF’ [MACS Neuro Medium (Miltenyi),l -glutamine 0.5 mM (Sigma), 1% ITS (Gibco), 1% AAS (Sigma-Aldrich)], ‘OLIGO’ [DMEM Glutamax high glucose (Gibco), 1% ITS (Gibco) 1% AAS (Sigma-Aldrich)], or ‘OLIGO + T3’ [DMEM Glutamax high glucose (Gibco), 1% ITS (Gibco) 1% AAS (Sigma-Aldrich) + T3 40 ng/ml (triiodothyronine, Sigma)].
For a complete list of reagents and materials used for cell culture, see Supplementary Table 1.
In order to evaluate the effect of different compositions of culture medium on OPCs differentiation, cells obtained with the MB method after 2 days in culture were then cultured in different media: ‘PROLIF – PDGFR-AA and FGF’ [MACS Neuro Medium (Miltenyi),
For a complete list of reagents and materials used for cell culture, see Supplementary Table 1.
5
Immortalized Human Mammary Fibroblasts
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Human mammary fibroblasts were extracted from a healthy breast tissue specimen that had been obtained by reduction mammoplasty prior to primary culture and immortalization with human telomerase reverse transcriptase as described previously.9 Human breast exp‐CAF2 cells and the corresponding control human mammary fibroblasts were also employed.9 These cells were cultured in DMEM high glucose GlutaMAX™ (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% Pen Strep (100 U/mL penicillin and 100 μg/mL streptomycin) (Gibco). MCF10DCIS.com (DCIS) cells were purchased from Asterand Bioscience. MDA‐MB‐231 cells were purchased from American Type Culture Collection. These breast cancer cells were cultured in DMEM/F‐12, GlutaMAX™ (Gibco) supplemented with 1% PenStrep (Gibco) with 5% FBS (DCIS cells) or 10% FBS (MDA‐MB‐231 cells).
6
Culturing GalT-GFP-SNAP26, hTERT-RPE1, and WISH Cells
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All cells were grown at 37 °C under 5% CO2 and routinely tested for the mycoplasma contamination. HeLa cells stably expressing GalT-GFP-SNAP26 (link) were cultured in DMEM high-glucose Glutamax (Gibco, Life Technologies), supplemented with 10% FCS (v/v) (Gibco, Life Technologies), 5 mM pyruvate (v/v) (Gibco, Life Technologies) and 1% penicillin–streptomycin (v/v) (Gibco, Life Technologies). hTERT-RPE1 (human retinal pigmented epithelial) cells (kind gift of P. Benaroch) were cultured in DMEM/F12 Glutamax (Gibco, Life Technologies), supplemented with 10% FCS. WISH cells (kind gift of D. Novick) were grown in MEM GlutaMAX (Gibco, Life Technologies) supplemented with 10% FCS, 5 mM pyruvate and 1% penicillin–streptomycin. WISH cells, which remain one of the most used cells in the IFN field, allowed to confirm and extend the role of the retromer on JAK/STAT signalling in another cell type. All cell lines were routinely tested for mycoplasma contamination.
7
Chondrogenic Differentiation of MSCs
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After expansion, MSCs were harvested and centrifuged at 200 × g for 8 min to obtain pellets of 200,000 cells. Chondrogenic differentiation was induced by culturing the cells for 5 weeks in chondrogenic medium consisting of DMEM-high-glucose GlutaMAX+ (GIBCO), 1:100 insulin, transferrin, and selenous acid (ITS+; BD Biosciences), 40 μg ml−1 L-proline (Sigma-Aldrich), 1 mM sodium pyruvate (GIBCO), 100 nM dexamethasone (Sigma-Aldrich), 10 ng ml−1 transforming growth factor β1 (TGF-β1; R&D Systems), 1.5 μg ml−1 fungizone, and 50 μg ml−1 gentamicin. Medium was renewed twice a week. Alternatively, the WNT inhibitor IWP2 (2 μM) was added during the last 3 weeks of chondrogenic induction. For this experiment, medium was renewed three times per week.
8
Protective Effects of PAD Inhibition in Neural Stem Cells
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Human tissues were supplied by Human Developmental Biology Resources under ethical approval. Human neural stem cell lines (hNSCs) from either the brain or the spinal cord of embryos between Carnegie stage (CS) 18 (gestation age 37–42 days) and CS22 (gestational age 54–56 days) were established and grown as previously described [36] (link). HEK293T (Human Embryonic Kidney 293) cells were grown DMEM–High Glucose–GlutaMAX (Gibco) supplemented with 10% fetal calf serum. The PAD inhibitor Cl-amidine [37] (link) was dissolved in phosphate buffer saline (PBS) and used at different concentrations (0.1–500 μM final concentration). Thapsigargin (Sigma) dissolved in ethanol was used at different concentrations (1–25 μM final concentration). Cells were treated with Cl-amidine 15 min before the addition of Thapsigargin. In some experiments the inhibitory effect of Cl-amidine treatment 15 min after the addition of Thapsigargin was also assessed.
9
Myoblast Culture and Differentiation
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All cells were grown at 37 °C under 5% of CO2. All myoblasts cell lines were cultured in Skeletal Muscle Cell Growth Medium (Promocell) supplemented with 20% FCS (Gibco, Life Technologies), 50 µg mL−1 of fetuine, 10 ng mL−1 of epidermal growth factor, 1 ng mL−1 basic fibroblast growth factor, 10 µg mL−1 of insulin, and 0.4 µg mL−1 of dexamethasone (Promocell). Prior to any cell seeding, surfaces (well, coverslip, patterned coverslips) are coated with 1% of matrigel (v/v) (Sigma) for 15 min at 37 °C. For myoblast differentiation, confluent cells (80–100% confluency) are put in DMEM high-glucose Glutamax (Gibco, Life Technologies), supplemented with 0.1% of insulin (v/v) (Sigma) for 4 days.
10
Lentiviral Vector-Based Umbilical Cord MSC Isolation
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The lentiviral vector plasmids were a gift from Tronolab (The EPFL University). The monoclonal antibodies against CD45, CD105, CD34, and CD44 were purchased from sigma (St. Louis, MO). The Mega Prep. Plasmid extraction kit was obtained from Macherey-Nagel & Co.KG (Germany). DMEM high glucose GlutaMAX™ and fetal bovine serum (FBS) were obtained from Gibco (USA). Escherichia coli (DH5α) was used for plasmid extraction. The 293LTV cell line used for the production of lentiviral particles was purchased from Iran’s Pasteur Institute (Tehran, Iran). With the informed consent and permission from the local ethics committee at Shiraz University of Medical Sciences, the umbilical cords (n=4) were obtained from full-term consenting caesarean patients at Ghadir Mother and Child Hospital (Shiraz, Iran), in sterile conditions. The adult male albino rats (n=24) were purchased from Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences (Shiraz, Iran).
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