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Dmem complete medium

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DMEM (Dulbecco's Modified Eagle Medium) is a complete cell culture medium that provides all the necessary nutrients for the growth and maintenance of a wide variety of cell types. It is a standardized formulation that includes amino acids, vitamins, inorganic salts, and other components required for optimal cell performance.

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Lab products found in correlation

Transit lt1, by Mirus Bio (3 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Puromycin, by InvivoGen (1 mentions) Penicillin, by Corning (1 mentions) Streptomycin, by Corning (1 mentions) L glutamine, by Corning (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Protease and phosphatase inhibitors tablets, by Thermo Fisher Scientific (1 mentions) Irdye 680rd goat anti rabbit igg secondary antibody, by LI COR (1 mentions) Irdye 800cw goat anti mouse igg secondary antibody, by LI COR (1 mentions) Immobilon fl 26 pvdf membrane, by Merck Group (1 mentions) Flag m2, by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Sk mes 1, by Thermo Fisher Scientific (1 mentions) Sk mes 1, by American Type Culture Collection (1 mentions)

8 protocols using dmem complete medium

1

Stable HEK293 cell lines expressing P2X7 and CD14

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HEK293 cells were cultured with DMEM complete medium (CORNING cellgro) and transfected with pLenti-GIII-CMV-hP2RX7-HA (Applied Biological Materials Inc.) or pcDNA3-hCD14 (Addgene) by using Lipofectamine 2000 (Invitrogen). The cells were positively selected by 1 mg/ml of G418 (Gemini Bio-Products) and 5 μg/ml of Puromycin (InvivoGen, CA, USA). For co-expressing cells, hP2X7R stably expressing HEK293 cells were transfected with pcDNA3-hCD14 and selected with neomycin.
Dagvadorj J., Shimada K., Chen S., Jones H.D., Tumurkhuu G., Zhang W., Wawrowsky K.A., Crother T.R, & Arditi M. (2015). Lipopolysaccharide induces alveolar macrophage necrosis via CD14 and the P2x7 receptor leading to Interleukin-1α release. Immunity, 42(4), 640-653.
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2

HepG2-CD81 Cell Line Maintenance

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HepG2-CD81 cells (Silvie et al., 2006 (link)) were modified from HepG2 hepatoma cells isolated from a hepatocellular carcinoma of a 15-year-old, Caucasian, male (ATCC, USA). Cells were maintained in DMEM-Complete Medium (Dulbecco’s modified eagle medium (Cellgro, Manassas, VA), supplemented with 10% v/v FBS (Sigma-Aldrich, St. Louis, MO), 10000 IU/ml penicillin, 100 mg/ml streptomycin (Cellgro), 2.5 mg/ml fungizone (HyClone/Thermo Fisher, Waltham, MA) and 4 mM L-Glutamine (Cellgro). Cells were split 2–3 times weekly.
Vijayan K., Arang N., Wei L., Morrison R., Geiger R., Parks K.R., Lewis A.J., Mast F.D., Douglass A.N., Kain H.S., Aitchison J.D., Johnson J.S., Aderem A, & Kaushansky A. (2022). A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection. Cell chemical biology.
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3

HEK 293T Cell Culture and Western Blot

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HEK 293 T cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO2 in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37 °C. Plasmid transfections were done with TransIT-LT1 (Mirus Bio) per the manufacturer’s instructions. Briefly, cell extracts were generated on ice in EBC buffer, 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP40, 1 mM DTT, and protease and phosphatase inhibitors tablets (Thermo Fisher Scientific). Extracted proteins were quantified using the PierceTM BCA Protein assay kit (Thermo Fisher). Proteins were separated by SDS acrylamide gel electrophoresis and transferred to IMMOBILON-FL 26 PVDF membrane (Millipore) probed with the indicated antibodies and visualized either by chemiluminescence (according to the manufacturer’s instructions) or using a LiCor Odyssey infrared imaging system. Western blot was conducted as described previously10 (link). Primary antibodies used for western blot are HA (Cat# 902302; 1:1000 dilution) from Biolegend and M2 FLAG (Cat# F1804; 1:1000 dilution) antibody from Sigma. Secondary antibodies used are IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LiCor) and IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LiCor). The study used a primary antibody against β-actin (Cat# A1978 from Sigma) for internal protein control.
Yuan G., Lu H., De K., Hassan M.M., Liu Y., Islam M.T., Muchero W., Tuskan G.A, & Yang X. (2023). Split selectable marker systems utilizing inteins facilitate gene stacking in plants. Communications Biology, 6, 567.
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4

Production and Characterization of ZIKV Replicon Virus Particles

Cited 2 times
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ZIKV RVPs were produced by modifying the DENV RVP system described previously [22 (link)–24 (link)]. An expression plasmid containing the CprM/E structural genes for ZIKV (strain SPH2015) was transfected into stable cell line BHK-DRRZ [28 (link)], which expresses the full-length DENV2 replicon with the CprM/E genes replaced by a gene for Renilla luciferase. After 72h, supernatants containing RVPs were harvested, passed through 0.45 μm filters, aliquoted, and stored at −80°C. For all experiments, frozen ZIKV RVPs (stored at −80°C) were thawed for 3 minutes in a 37°C water bath and then placed on ice before use. The following cell lines were used in this study: K562, BHK, HEK-293T, Vero (all from ATCC), Raji-DC-SIGN-R (kindly provided by Robert Doms), BHK-DRRZ cells expressing DENV2 replicon, and BHK cells expressing DC-SIGN (derived from BHK21 clone 15, Center for Vector-borne Diseases, UC Davis). All cell lines were grown at 37°C at 5% CO2 in DMEM complete medium (DMEM with 10% FBS and 1% penicillin-streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% MEM non-essential amino acids) (Corning). Raji-DC-SIGN-R cells were grown in RPMI with 10% FBS and 1% penicillin-streptomycin and 2 mM L-glutamine.
Whitbeck J.C., Thomas A., Kadash-Edmondson K., Grinyo-Escuer A., Stafford L.J., Cheng C., Liao G.C., Holtsberg F.W., Aman M.J., Simmons G., Davidson E, & Doranz B.J. (2020). Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications. PLoS Neglected Tropical Diseases, 14(11), e0008730.
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5

Cell Culture and Transfection Protocol

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HeLa, HEK 293T, and Glioblastoma U87 cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO2 in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37 °C. Plasmid transfections were done with TransIT-LT1 (Mirus Bio) per the manufacturer’s instructions.
Pal D., De K., Shanks C.M., Feng K., Yates T.B., Morrell-Falvey J., Davidson R.B., Parks J.M, & Muchero W. (2022). Core cysteine residues in the Plasminogen-Apple-Nematode (PAN) domain are critical for HGF/c-MET signaling. Communications Biology, 5, 646.
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6

Culturing and Transfecting Common Cell Lines

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Vero-E6 cells were obtained from Carmen Foster at the ORNL as a generous gift. HeLa and HEK 293T cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO2 in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37°C. Plasmid transfections were done with TransIT-LT1 (Mirus Bio) per the manufacturer’s instructions. Detailed information regarding all the cell lines used was provided in the “key resources table.”
Pal D., De K., Yates T.B., Kolape J, & Muchero W. (2023). Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization. iScience, 26(4), 106274.
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7

Culturing Adenocarcinoma and Squamous Carcinoma Cell Lines

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The adenocarcinoma cell lines A549 and PC-9, squamous carcinoma cell line SK-MES-1 were purchased from the American Type Culture Collection (ATCC), and maintained at 37˚C under 5% CO2. A549 and PC-9 cultured in complete DMEM medium (Corning, NY, USA). SK-MES-1 cultured in complete MEM medium with 1% 100mM sodium pyruvate (Gibco). All complete medium contained 10% heat-inactivated fetal bovine serum (Gibco, USA).
Zhang R., Dong Y., Sun M., Wang Y., Cai C., Zeng Y., Wu Y, & Zhao Q. (2019). Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway. Journal of Cancer, 10(4), 1004-1012.
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8

Quantifying Arrestin-Mediated Receptor Interactions

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BRET-based [27 (link), 28 (link)] protein-protein interaction assays were performed to assess the arrestin-mediated interaction of JNK3α2 with angiotensin II receptor type I (AT1R) following the procedure used for the arrestin-receptor interaction assay [26 (link), 29 (link), 30 ]. Cultured COS-7 cells were reseeded into 60 mm dishes 24 h before transfection in complete DMEM medium (Mediatech-Corning, Manassas, VA). Plated cells were transfected with Lipofectamine 2000 (Life Technologies) following the manufacturer’s instructions with AT1R-RLuc8 (AT1R fused to Renilla luciferase variant 8 at the C-terminus) coding plasmid (0.6 μg/dish), bovine arrestin-3 (0–6 μg/dish) and Venus N-terminus-tagged JNK3α2 (3 μg/dish). Cells were transferred to white opaque 96 well plates and luminescence at 460 and 535 nm was measured 48 h post-transfection. Cells were stimulated by 1 μM angiotensin II (agonist) or vehicle (control) followed by the addition of 5 μM coelenterazine-h (Renilla luciferase substrate). Measurements were taken at 10-minute intervals for up to 90 minutes. Angiotensin-induced net BRET was determined by subtracting the 535 nm/460 nm BRET ratio in the absence of agonist from the ratio of agonist-treated cells for each arrestin concentration. Results are expressed as the net BRET values for each amount of arrestin-3 at a given time point after agonist stimulation.
Zhan X., Perez A., Gimenez L.E., Vishnivetskiy S.A, & Gurevich V.V. (2014). Arrestin-3 binds the MAP kinase JNK3α2 via multiple sites on both domains. Cellular signalling, 26(4), 766-776.
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