PC12 cells (CRL-1721; ATCC; passages < 10) were cultured in Dulbecco’s modified eagle medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), penicillin (100 units/mL), and streptomycin (100 µg/mL), under a humidified atmosphere containing 5% CO2 at 37 °C. Except for morphology test, cells were exposed to 50 ng/mL of nerve growth factor (NGF; Sigma-Aldrich, St. Louis, MO, USA) dissolved in DMEM medium containing 1% FBS and HS for 48 h to differentiate. All agents applied in cell culture were purchased from Invitrogen, Carlsbad, CA, USA.
Dulbecco s modified eagle medium
Manufactured by American Type Culture Collection
Sourced in United States
Dulbecco's modified Eagle medium (DMEM) is a cell culture medium that provides essential nutrients and growth factors for the cultivation of various cell types. It is a widely used medium in cell biology and biomedical research. DMEM is designed to support the growth and maintenance of cells in vitro.
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Lab products found in correlation
Fetal bovine serum (fbs), by American Type Culture Collection (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by American Type Culture Collection (3 mentions)
Spin desalting column, by Thermo Fisher Scientific (2 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Plv mcherry, by Addgene (2 mentions)
Phoenix cells, by American Type Culture Collection (2 mentions)
Human dermal microvascular endothelial cells hdmvecs, by ScienCell (2 mentions)
Endothelial cell culture medium, by ScienCell (2 mentions)
C57bl 6j wild type and ldl receptor ldlr ko b6.129s7 ldlrtm1her j mice, by Jackson ImmunoResearch (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Penicillin, by American Type Culture Collection (2 mentions)
Human arpe 19 cells, by American Type Culture Collection (2 mentions)
Wm266 4, by American Type Culture Collection (1 mentions)
Wm115, by American Type Culture Collection (1 mentions)
Hs895 sk, by American Type Culture Collection (1 mentions)
Hs895 t, by American Type Culture Collection (1 mentions)
Te353 sk, by American Type Culture Collection (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
24 protocols using dulbecco s modified eagle medium
1
PC12 Cell Differentiation with NGF
2
Comparative Imaging of Isogenic Cell Lines
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We imaged and analyzed four isogenic cell lines, termed SW28 (link),29 (link), HS, TE, and WM. The SW cells are a pair of isogenic cancer cell lines: SW480 (ATCC CCL-228)—colorectal adenocarcinoma cells, and SW620 (ATCC CCL-227)—a metastatic form of these cancer cells collected from a lymph node of the same patient. The HS cells are a pair of a normal skin cell line, HS895.Sk (ATCC CRL-7636) and a melanoma cancer cell line, HS895.T (ATCC CRL-7637), taken from the same patient. The TE cells are a pair of a normal skin cell line, TE353.Sk (ATCC CRL-7761), and a melanoma cancer cell line, TE354.T (ATCC CRL-7762), are both taken from the same patient. The WM cells are a pair of a melanoma skin cell line, WM115 (ATCC CRL-1675), and a metastatic melanoma skin cell line, WM266.4 (ATCC CRL-1676), both taken from the same patient.
All the cells were grown in Dulbecco's modified Eagle medium (ATCC, SN. 30-2002), supplemented with 10% fetal bovine serum (BI, SN. 04-007-1A). 2 mM L-glutamine (BI, SN. 03-020-1B) were added for the WM cell lines. The cells were incubated under standard humidity and temperature of 37 °C with 5% CO2 until 80% confluence was reached. Before imaging, the cells were trypsinized for suspension and supplemented with a suitable medium.
All the cells were grown in Dulbecco's modified Eagle medium (ATCC, SN. 30-2002), supplemented with 10% fetal bovine serum (BI, SN. 04-007-1A). 2 mM L-glutamine (BI, SN. 03-020-1B) were added for the WM cell lines. The cells were incubated under standard humidity and temperature of 37 °C with 5% CO2 until 80% confluence was reached. Before imaging, the cells were trypsinized for suspension and supplemented with a suitable medium.
3
SARS-CoV-2 Spike Protein Internalization in Endothelial Cells
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Human dermal microvascular endothelial cells (hDMVECs) and endothelial cell (EC) culture medium were purchased from ScienCell (San Diego, CA, USA). Phoenix cells and Dulbecco's Modified Eagle Medium (DMEM) were purchased from ATCC (Manassas, VA, USA). Lentiviral vector pLV-mCherry were obtained from Addgene (Watertown, MA, USA). Coding sequence of SARS-CoV-2 Spike gene (Wuhan variant, GenBank: QHU36824.1 ) and the expression vector were described previously [22 (link)]. LDL-c, HDL-c and lipoprotein depleted fetal bovine serum (LD-FBS) were obtained from Kalen Biomedical, LLC (Germantown, MD, USA). Sulfo-NHS-LC-biotin and desalting spin column were obtained from ThermoFisher (Waltham, MA, USA). The sources of antibodies were listed in the supplementary table 1 . Primers were synthesized by IDT (Coralville, IA, USA) and listed in supplementary table 2 . The animal protocol was approved by the IACUC committee at the University of South Carolina, Columbia. C57BL/6J wild type and LDL receptor (LDLR) KO (B6.129S7-Ldlrtm1her/J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The generation of Spp virus was previously reported [22 (link)].
4
SARS-CoV-2 Spike Protein Transduction in Endothelial Cells
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Human dermal microvascular endothelial cells (hDMVECs) and endothelial cell (EC) culture medium were purchased from ScienCell (San Diego, CA, USA). Phoenix cells and Dulbecco's Modified Eagle Medium (DMEM) were purchased from ATCC (Manassas, VA, USA). Lentiviral vector pLV-mCherry were obtained from Addgene (Watertown, MA, USA). Coding sequence of SARS-CoV-2 Spike gene (Wuhan variant, GenBank: QHU36824.1) and the expression vector were described previously [22 (link)]. LDL-c, HDL-c and lipoprotein depleted fetal bovine serum (LD-FBS) were obtained from Kalen Biomedical, LLC (Germantown, MD, USA). Sulfo-NHS-LC-biotin and desalting spin column were obtained from ThermoFisher (Waltham, MA, USA). The sources of antibodies were listed in the Supplementary Table 1 . Primers were synthesized by IDT (Coralville, IA, USA) and listed in Supplementary Table 2 . The animal protocol was approved by the IACUC committee at the University of South Carolina, Columbia. C57BL/6J wild type and LDL receptor (LDLR) KO (B6.129S7-Ldlrtm1her/J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The generation of Spp virus was previously reported [22 (link)].
5
Culturing Adult Human Dermal Fibroblasts
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Commercial human dermal fibroblasts (NHDF adult, Promocell, Heidelberg, Germany) were used for all cell experiments. NHDF fibroblasts were cultured in medium containing 72 % Dulbecco’s modified Eagle medium (ATCC, Manassas, USA), 18 % Medium M199 (Sigma-Aldrich, Steinheim, Germany), 9 % fetal calf serum (PAA Laboratories, Pasching, Austria) and 1 % Penicillin–Streptomycin (Invitrogen, Karlsruhe, Germany). Cells were incubated at 37 °C in humidified air with 5 % CO2 atmosphere. Culture medium was changed twice a week. Cells were subcultured by detachment with Accutase (PAA Laboratories, Pasching, Austria) at approximately 90 % confluence. Fibroblasts from the 2nd to 6th passage were used for all experiments.
6
Caco-2 Cell Culture and EV Isolation
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Caco-2 HTB-37 (ATCC, USA),
FHs 74 Int (ATCC, USA), streptomycin/penicillin solution (Gibco, USA),
phosphate-buffered saline (PBS) (Gibco, USA), and trypsin/EDTA (Gibco,
USA), β-Cyclodextrin (Sigma Aldrich, USA), Dulbecco’s
modified eagle medium (ATCC, USA), fetal bovine serum (Gibco, USA),
and EV (Toronto Research Chemicals, Canada).
FHs 74 Int (ATCC, USA), streptomycin/penicillin solution (Gibco, USA),
phosphate-buffered saline (PBS) (Gibco, USA), and trypsin/EDTA (Gibco,
USA), β-Cyclodextrin (Sigma Aldrich, USA), Dulbecco’s
modified eagle medium (ATCC, USA), fetal bovine serum (Gibco, USA),
and EV (Toronto Research Chemicals, Canada).
7
Glioma Cell Line Cultivation and Characterization
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Materials for tissue culture were purchased either from Euroclone Italia (Euroclone S.p.A, Milan, Italy) or from ATCC (Manassas, VA, USA). Three human glioma cell lines, U251(EATCC; 09063001), U87MG (ATCC; HTB-14), and T98G (ATCC; CRL190), were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) containing 10% (v/v) fetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1% nonessential amino acids (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). The risk of working with misidentified and/or contaminated cell lines was minimized by using GBM cells at very low passages and periodic short tandem repeat (STR) DNA profiling. Luciferase-tagged U87MG (U87MG-Luc) cells were generated and provided by Jari E. Heikkila (Abo Akademi University, Turku, Finland). A GBM patient-derived stem cell (GSC) lines (BT48EF and CSCs-5) were provided by J. Gregory Cairncross and Samuel Weiss (University of Calgary, Canada) and by M. Izquierdo (Universidad Autónoma de Madrid, Spain), respectively (Luchman et al., 2012 (link); Mendiburu-Elicabe et al., 2014 (link)). Isolated neurospheres of U87 and GSC cells were assayed for ‘stemness’ properties in terms of clonogenic capacity and positivity for established stem cell markers (Gravina et al., 2019 (link)).
8
Cell Culture Media Preparation
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RPMI 1640 medium, Dulbecco’s Modified Eagle Medium and F-12K medium were purchased from ATCC (American Type Culture Collection, from Rockville, MD, USA). All chemicals were purchased from Sigma–Aldrich, Darmstadt, Germany.
9
Cellular and Material Characterization
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RAW264.7 cell, ARPE-19 cell, MOVAS cell, SVEC, and fetal bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco's modified eagle medium (DMEM) were all purchased from ATCC (Manassas, VA). L-929 cell and mouse primary tenocyte (kindly provided by Dr. Rupak Rajachar, Houghton, MI). Silanol-terminated PDMS, (3-aminopropyl) trimethoxylsilane, dibutyltin dilaurate were purchased from Gelest Inc. (Morrisville, PA). Room temperature vulcanized PDMS (RTV-3140) and silicone elastomer base and curing agent (Sylgard® 184) were acquired from Dow Corning Co. (Midland, MI). Interferon-γ mouse was obtained from BD Biosciences (San Diego, CA), lipopolysaccharine, L-arginine, N-acetyl-D, L-penicillamine, acetyl anhydrate, calcein-AM, and 4-amino-5-methylamino-2′,7′-difluorescein (DAF-FM), and cyclam were obtained from Sigma-Aldrich (St. Louis, MO). Tertbutyl nitrite was purchased from Acros Organics, (Pittsburgh, PA). Toluene was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Nω-hydroxy-nor-Arginine (nor-NOHA), L-NG-Nitroarginine methyl ester, and nitrate/nitrite colorimetric assay kit were obtained from Cayman Chemical (Ann Arbor, MI). Gelatin was obtained from Bio-rad (Hercules, CA). Pyridine was purchased from EMD Chemical Inc. (Darmstadt, Germany). Ethidium bromide was obtained from Invitrogen (Grand Island, NY).
10
VEEV TC-83 Virus Propagation in BHK Cells
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Vero 76 cells (ATCC CRL-1587) were maintained at 37°C and 5% CO2 in complete medium containing Dulbecco’s modified Eagle medium with Hi-glucose and L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. VEEV TC-83 was obtained from Connie Schmaljohn (United States Army Medical Research Institute of Infectious Diseases, Maryland). Virus seed stocks were amplified in Bovine hamster kidney (BHK)cells in minimum essential medium with Earle’s salts Eagle's minimum essential medium (EMEM) with 2% FBS and 1% penicillin-streptomycin. All cell culture reagents were purchased from Thermo Fisher Scientific unless otherwise specified. VEEV TC-83 was sequenced as a reference genome as previously reported (34 (link)).
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