Dulbecco modified eagle medium (dmem)

Manufactured by InvivoGen

Sourced in United States, France

DMEM (Dulbecco's Modified Eagle's Medium) is a basal medium commonly used for the in vitro culture of various cell types. It provides the necessary nutrients, vitamins, and salts required for cell growth and maintenance.

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Lab products found in correlation

Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (15 mentions) Blasticidin, by InvivoGen (6 mentions) Normocin, by InvivoGen (5 mentions) Penicillin, by Thermo Fisher Scientific (5 mentions) Penicillin, by InvivoGen (4 mentions) Streptomycin, by InvivoGen (4 mentions) Zeocin, by InvivoGen (3 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Hygromycin, by InvivoGen (2 mentions) Penicillin streptomycin, by InvivoGen (2 mentions) Penicillin, by PAN Biotech (2 mentions) Streptomycin, by PAN Biotech (2 mentions) Hek293t cell, by American Type Culture Collection (2 mentions) U937 cells, by American Type Culture Collection (1 mentions) Lps ek, by InvivoGen (1 mentions) Streptomycin p s, by Thermo Fisher Scientific (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)

25 protocols using dulbecco modified eagle medium (dmem)

Cell Line Establishment and Characterization

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Human LPS cell lines Lipo224, Lipo246, Lipo863 were established in our laboratory as previously reported (11 (link)). LPS141 was kindly provided by Dr. Jonathan Fletcher (Brigham and Women’s Hospital). LPS cells were maintained using standard conditions and were grown in DMEM (Gibco), supplemented with 10% (vol/vol) FBS. U937 cells (ATCC) were cultured in RPMI (Gibco), supplemented with 10% (vol/vol) FBS. For differentiation, cells were incubated with 12-myristate 13-acetate (PMA) 10 ng/ml for 24h. Human HEK-Blue-293 (Invivogen) cells were cultured in DMEM supplemented with 10% (vol/vol) FBS, Normocin (50 μg/mL), Blasticidin (10 μg/mL), and Zeocin (100 μg/mL) (Invivogen). Preadipocytes (XA15A1) were purchased from Lonza and maintained following the manufacturer’s instructions. Murine Lewis lung carcinoma cells (LLC) (ATCC) were cultured in RPMI 1640, supplemented with 10% (vol/vol) FBS.
All the cell line used in this study were acquired within the past 5 years and authenticated by STR on 2/9/2017. Murine Lewis lung carcinoma cells (LLC) were authenticated by morphology and biologic behavior. Preadipocytes and HEKBlue 293 were bought within 6 month from this manuscript submission. Lonza tested the cells for differentiation, and stained for adipocytes; HEK-Blue-293 were thoroughly tested and validated by InvivoGen. All cell lines were tested for mycoplasma.

Casadei L., Calore F., Creighton C.J., Guescini M., Batte K., Iwenofu O.H., Zewdu A., Braggio D.A., Lynn Bill K., Fadda P., Lovat F., Lopez G., Gasparini P., Chen J.L., Kladney R.D., Leone G., Lev D., Croce C.M, & Pollock R.E. (2017). Exosome-derived miR-25-3p and miR-92a-3p stimulate liposarcoma progression. Cancer research, 77(14), 3846-3856.

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Astrocyte Stimulation with LPS

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Purified primary murine astrocytes were seeded onto 12-well plates at a concentration of 1 × 10⁴ cells/cm² in 10% bovine calf serum (BCS; HyClone, Logan, UT)/DMEM (v/v). After an initial 24-h incubation at 37 °C in 5% CO₂/95% humidified air, the medium was removed and the cultures were washed once with PBS (pH 7.4) to remove cell debris and non-adherent cells. Next, 10% BCS/DMEM was added to adherent cells (2 mL), and the astrocytes were allowed to proliferate at 37 °C in 5% CO₂/95% humidified air for 48 h. Following this incubation, the supernatant was replaced with fresh 10% BCS/DMEM (2 mL in 12-well plates, 3 mL in 6-well plates) in the presence of varying concentrations of LPS from Escherichia coli K12 (LPS-EK: InvivoGen, San Diego, CA) dissolved in PBS (pH 7.4; PBS alone as a negative control). This step represented the start of the experiment (i.e., T₀).

Rodgers K.R., Lin Y., Langan T.J., Iwakura Y, & Chou R.C. (2020). Innate Immune Functions of Astrocytes are Dependent Upon Tumor Necrosis Factor-Alpha. Scientific Reports, 10, 7047.

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Dermal Fibroblast Poly(I:C) Response

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Human dermal fibroblasts (HDFs) were purchased from Gibco. Primary mouse dermal fibroblasts (MDFs) were prepared from neonatal mice skin as previously described [14 (link)]. Cells were cultured in Dulbecco’s Modified Essential Medium (DMEM, Welgene, Gyeongsan-si, Korea) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 units/mL penicillin and 100 µg/mL streptomycin (PS) (Thermo Fisher Scientific, Waltham, MA, USA). For poly(I:C) (invivogen, San Diego, CA, USA, tlrl-pic-5) treatment, cells were first serum-starved starved for 24 h in DMEM containing 0.1% FBS. Then, cells were treated with saline (for vehicle) or indicated concentrations of poly(I:C) for 12 or 24 h. MDFs or HDFs were used under passage 3 or 8, respectively.

Hong A.Y., Lee S.J., Lee K.B., Shin J.W., Jeong E.M, & Kim I.G. (2022). Double-Stranded RNA Enhances Matrix Metalloproteinase-1 and -13 Expressions through TLR3-Dependent Activation of Transglutaminase 2 in Dermal Fibroblasts. International Journal of Molecular Sciences, 23(5), 2709.

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Cell Culture Protocols for Immune Studies

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All media described below were supplemented with 10% fetal bovine serum (Sigma-Aldrich). Cell cultures included the following: human middle ear epithelial cell (HMEEC)-1 cells (provided by Dr. David J. Lim (35 (link))) were maintained in DMEM (Corning Cellgro) and supplemented with bronchial epithelial cell growth medium SingleQuots (Lonza), HEK293-TLR4/MD2 stably-expressing cells (provided by Dr. Douglas T. Golenbock (36 (link))) in DMEM supplemented with G418 (InvivoGen) (19 (link)), HeLa cells (American Type Culture Collection, no. CCL-2) in DMEM, BEAS-2B cells (American Type Culture Collection, no. CRL-9609) in RPMI 1640 medium (Life Technologies), and A549 cells (American Type Culture Collection, no. CCL-185) in F-12K medium (Life Technologies). Mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) or TLR4^−/− mice (provided by Dr. Shizuo Akira) and maintained in DMEM media. All cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C.

Matsuyama S., Komatsu K., Lee B.C., Tasaki Y., Miyata M., Xu H., Shuto T., Kai H, & Li J.D. (2022). Negative cross-talk between TLR2/4-independent AMPKα1 and TLR2/4-dependent JNK regulates S. pneumoniae-induced mucosal innate immune response. Journal of immunology (Baltimore, Md. : 1950), 209(8), 1532-1544.

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Culturing HEK293T and iSLK.RTA cells

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HEK293T cells were cultured at 37 °C, 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, GE Healthcare, Chicago, IL, USA). iSLK.RTA cells (kind gift of Dr. Jung) containing wild-type KSHV BAC16, BAC16 ORF42^PTC, BAC16 ORF42^REV, or BAC16 ORF42-Flag were maintained in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin (Enzo Lifesciences, New York, NY, USA). KSHV-infected iSLK.RTA cells that also express a transgene (ORF42^PTC, +ORF42, +ORF42-Flag and +empty) were maintained in DMEM supplemented with 10% FBS, 400 μg/mL hygromycin and 100 μg/mL zeocin (Invivogen, San Diego, CA, USA). Live cells were imaged using a Nikon eclipse TE2000-U.

Butnaru M, & Gaglia M.M. (2019). The Kaposi’s Sarcoma-Associated Herpesvirus Protein ORF42 Is Required for Efficient Virion Production and Expression of Viral Proteins. Viruses, 11(8), 711.

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High-Throughput Screening of Immune Modulators

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2,296 molecules were screened (Supplementary Table 1) and derived from the rolling and curated ICCB-Longwood Screening Facility at Harvard Medical School. All plates used a 384 well format. 704 molecules were chosen for known activity toward PBMCs; these molecules filled up two library plates and originated from the ChemDiv6 library (ChemDiv Inc, San Diego, CA). Another 1592 molecules were chosen from the Selleck bioactive chemical plates (Selleck Chemicals LLC, Houston, TX). The compounds (stored at 10 mM in dessicated conditions) were pinned at a volume of 100 nL, for a final concentration of 33 μM in a 384 well format. 5 μL of 0.3% DMSO(Millipore, Burlington, MA) diluted in DMEM, the negative control, and the TLR9 agonist ODN2395 and the TLR7/8 agonist R848 (both from Invivogen, San Diego, CA), the positive controls, were added to the wells manually for final concentrations of 1μM and 25 μM respectively. PBMCs were then stimulated for 72 hours at 37°C, 5% CO₂ in a humidified ThermoScientific Forma CO₂ Incubator (Waltham, MA).

Chew K., Lee B., van Haren S.D., Nanishi E., O’Meara T., Splaine J.B., DeLeon M., Soni D., Seo H.S., Dhe-Paganon S., Ozonoff A., Smith J.A., Levy O, & Dowling D.J. (2022). Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells. bioRxiv.

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Murine Bone Marrow-Derived Macrophage Culture

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BMDMs were cultured using the protocol originally described by Weischenfeldt and Porse [44 ] with slight modification [20 (link)]. Briefly, tibias and femurs were collected from 8 to 12-week old wild-type C57BL/6J mice. Bone marrow was flushed with DMEM (Gibco BRL, Paisley, UK). Red Blood Cells (RBCs) were lysed by RBC Lysis Buffer (0.75% NH₄Cl, 0.02% Tris-HCl, pH 7.2). Cells were then washed and cultured in DMEM supplemented with 15% FCS and 15% L929 (a murine fibroblast cell line that secretes M-CSF) conditioned medium containing 100 mg/ml primocin (Invivogen, San Diego, California, USA) in 75 cm² Culture Flasks, and incubated at 37°C in 5% CO₂ incubator. After 6 days, cells were collected for further experiments. Flow cytometry confirmed that >92% of the cells were F4/80⁺CD11b⁺.

Luo C., Zhao J., Chen M, & Xu H. (2018). The expression of C1 inhibitor (C1INH) in macrophages is upregulated by retinal pigment epithelial cells – implication in subretinal immune privilege in the aging eye. Aging (Albany NY), 10(6), 1380-1389.

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Cell Culture and Proliferation Analysis

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Adherent or suspension cells were cultured in DMEM or RPMI (ThermoFisher Scientific), respectively, supplemented with 10–15% fetal calf serum (FCS, Life Technologies), ultraglutamine as needed, and antibiotics. Mouse embryo and human foreskin fibroblasts were immortalized using an HIV-based vector encoding hTERT, the catalytic subunit of human telomerase, or SV40 virus large T antigen, coupled by an internal ribosome entry site to bsd, and maintained in DMEM supplemented with 10 μg/ml blasticidin (Invivogen).
For cell cycle analysis, mid-logarithmic cells were lifted in PBS plus 2 mM EDTA, pelleted, resuspended in complete medium supplemented with 10 μg/mL Hoechst 33342, and incubated for 45 min at 37°C prior to data acquisition on an LSRII flow cytometer (B-D). Cell cycle analysis was performed using FlowJo software. Because MTT assay results were misleading due to the increased size of the UBXN1 KO cells, for proliferation studies cells were plated at low density in replicates in 6-well format and enumerated manually every 48 h after trypsinization, using a hemocytometer and trypan blue exclusion dye staining, passaging as necessary.

Hu Y., O’Boyle K., Auer J., Raju S., You F., Wang P., Fikrig E, & Sutton R.E. (2017). Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα. PLoS Pathogens, 13(2), e1006187.

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Cell Culture Protocols for Immune Studies

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HeLa cells (human cervical cancer cell line, Korea Cell Line Bank, South Korea) and HEK 293T cells (human embryonic kidney‐293T, ATCC, USA) and Raw 264.7 (mouse macrophage, ATCC, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (P/S, Gibco, USA). These cultures were maintained in a 5% CO₂ incubator at 37 °C. THP1‐Dual and THP1‐Dual KO cells (RIG‐I KO, MAVS KO, MDA5 KO, cGAS KO, STING KO cells, Invivogen, Hong‐Kong) were cultured in RPMI 1640 media (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin/streptomycin (P/S, Gibco, USA), and selective antibiotics. These cells were also maintained in a 5% CO₂ incubator at 37 °C, following the manufacturer's instructions. Raw‐Dual cells (Invivogen, Hong‐Kong) were cultured in DMEM supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin.

Son S., Park M., Kim J, & Lee K. (2024). ACE mRNA (Additional Chimeric Element incorporated IVT mRNA) for Enhancing Protein Expression by Modulating Immunogenicity. Advanced Science, 11(18), 2307541.

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Establishing Stable MDA5-Expressing Cell Line

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The stable cell line expressing MDA5 was obtained by transfecting HEK293 cells with the plasmid pUNO harboring full-length MDA5 (Invivogen, Toulouse, France). Monolayer of HEK293 cells (200 × 10³ cells) were transfected by mixing 1 µg of plasmid with 1 µL polyethylenimine (~Mw 25k, Sigma-Aldrich, St. Louis, MO, USA), then diluted in 50 µL of DMEM (Eurobio scientific, Les Ulis, France). The stably transfected HEK293 cells were selected and maintained in DMEM supplemented with 10% FBS, glutamine, antibiotics (penicillin and streptomycin), and the selection antibiotic blasticidin (20 μg/mL, Invivogen). In order to enrich the cell culture with positive cells, several rounds of cloning by limit dilution were conducted until the generation of more than 90% of stable cells expressing MDA5. Cells stably expressing MDA5 were then mixed with untransfected HEK293 cells in a 1:5 ratio and cultured in 96-well plates (1 × 10⁴ cell/well) for 48 h at 37 °C in order to easily discriminate, via IIF, the specific staining from the nonspecific background in each well.

Nombel A., Pin J.J., Fabien N., Miossec P, & Coutant F. (2022). Evaluation of the Performance of an Indirect Immunofluorescence Assay for the Detection of Anti-MDA5 Antibodies. Biomedicines, 10(11), 2969.

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