Dmem hepes

Manufactured by Thermo Fisher Scientific

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DMEM-HEPES is a cell culture medium formulation that contains the buffering agent HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). It is designed to maintain a stable pH in cell culture applications.

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HEPES (4332 citations) DMEM/F12 HEPES (12 citations) HEPES-buffered DMEM (11 citations) DMEM and HEPES 2.4% (2 citations) Dulbecco’s Modified Eagle Medium (DMEM/HEPES, (2 citations) DMEM/F-12 with HEPES (2 citations) Hepes buffered DMEM without phenol red (2 citations) DMEM)‐HEPES medium (2 citations) DMEM-F12 with HEPES (DMEM-F12/HEPES), (2 citations) DMEM 25 mM Hepes (1 citations)

17 protocols using dmem hepes

Maintenance of Cell Lines for Viral Transduction

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Ba/F3 cells were purchased from DSMZ and maintained in 90% RPMI (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 10ng/ml IL-3 (Fisher), and incubated at 37°C with 5% CO2. Ba/F3 cells were passaged at or below 1.0E6 cells/ml in order to avoid acquired IL-3 resistance, and regularly checked for IL-3 addiction by performing 3x PBS (Gibco) washes and outgrowth in the absence of IL-3 to confirm cell death in the parental, empty cell line.
Plat-E cells stably expressing retroviral envelope and packaging plasmids were originally gifted by Dr. Wendell Lim. Plat-E cells were maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), 10ug/ml blasticidin, 1ug/ml puromycin, and incubated at 37°C with 5% CO2. Plat-E cells were maintained under blasticidin and puromycin antibiotic pressure unless being transfected.
HEK293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in 5% CO2.
Human MET knockout HeLa cells were purchased from Abcam and maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and incubated at 37°C with 5% CO2

Estevam G.O., Linossi E.M., Macdonald C.B., Espinoza C.A., Michaud J.M., Coyote-Maestas W., Collisson E.A., Jura N, & Fraser J.S. (2023). Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain. bioRxiv.

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Culturing and Transfecting HEK293T Cells

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HEK293T cells (ATCC) were grown in DMEM + HEPES (Gibco) supplemented with 10% FCS [Sigma or Mediatech (Corning)], 1 × glutamax (Gibco), 1 × penicillin and streptomycin (Gibco) at 37 °C in the presence of 5% CO₂. All transfections were performed 24 h after cells were seeded using PEI (polyethylenimine) (Polysciences, USA). In all experiments the ratio of PEI:DNA was 3 μg:1 μg.

Rossi J.J., Rosenfeld J.A., Chan K.M., Streff H., Nankivell V., Peet D.J., Whitelaw M.L, & Bersten D.C. (2021). Molecular characterisation of rare loss-of-function NPAS3 and NPAS4 variants identified in individuals with neurodevelopmental disorders. Scientific Reports, 11, 6602.

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Cell Culture Protocols for Vero, C6/36, and Aag2 Cells

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African green monkey kidney Vero E6 (ATCC CRL-1586) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, CA, United States) containing 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/ml; Sigma-Aldrich, Saint Louis, MO, United States), and streptomycin (100 μg/ml; Sigma-Aldrich) (P/S). Vero cells were cultured as monolayers in T25 cell culture flasks (Greiner Bio-One, Kremsmünster, Austria) at 37°C with 5% CO₂, and split every 3–4 days. Prior to infections, Vero cells were seeded in DMEM containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (DMEM-HEPES; Gibco) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), hereafter named DMEM-supplemented. Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured in Leibovitz L-15 medium (Gibco) supplemented with 10% FBS, 2% tryptose phosphate broth (Gibco), and 1% nonessential amino acids (Gibco), hereafter named Leibovitz-complete. Aedes aegypti Aag2 cells were cultured in Schneider’s Drosophila medium (Lonza, Basel, Switzerland) supplemented with 10% FBS, hereafter named Schneider’s-complete. Both C6/36 and Aag2 cells were cultured as monolayers in T25 flasks at 28°C, and split every 3–4 days.

Göertz G.P., Vogels C.B., Geertsema C., Koenraadt C.J, & Pijlman G.P. (2017). Mosquito co-infection with Zika and chikungunya virus allows simultaneous transmission without affecting vector competence of Aedes aegypti. PLoS Neglected Tropical Diseases, 11(6), e0005654.

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HeLa Cell Culture and Infection

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HeLa cells (Supplementary file 2) were grown in DMEM supplemented with 300 μg/ml L-Glutamine, 100 U/ml Penicillin, 100 μg/ml Streptomycin and 10% FCS at 37°C and 5% CO₂. For infection experiments, HeLa cells were seeded in 24-well plates. When cultures reached ~10⁶ cells/well, they were washed twice with PBS and medium was changed to DMEM-HEPES (Gibco, United States) without supplements. HeLa cells were routinely tested for absence of mycoplasma contamination by EZ-PCR Mycoplasma Test Kit (Biological Industries LTD., Israel).

Ronin I., Katsowich N., Rosenshine I, & Balaban N.Q. (2017). A long-term epigenetic memory switch controls bacterial virulence bimodality. eLife, 6, e19599.

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Cell Culture for Arbovirus Research

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C6/36 (ATCC CRL-1660) and Culex tarsalis cells (CDC, Fort Collins, CO) were grown on Leibovitz L15 and Schneiders (Gibco) medium supplemented with 10% fetal bovine serum (FBS; Gibco). Hela (ATCC CCL-2), DF-1 (ATCC CRL-12203) and Vero E6 (ATCC CRL-1586) cells were cultured with DMEM Hepes (Gibco, Bleiswijk, The Netherlands) buffered medium supplemented with 10% FBS containing penicillin (100IU/ml) and streptomycin (100μg/ml). When Vero E6 cells were incubated with mosquito lysates or saliva the growth medium was supplemented with fungizone (2,5μg/ml) and gentamycin (50μg/ml). P2 virus stocks of the NY’99 and Gr’10 isolates were grown on C6/36 cells and titrated on Vero E6 cells.

Fros J.J., Geertsema C., Vogels C.B., Roosjen P.P., Failloux A.B., Vlak J.M., Koenraadt C.J., Takken W, & Pijlman G.P. (2015). West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance. PLoS Neglected Tropical Diseases, 9(7), e0003956.

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Fibrillar Collagen Gel Fabrication

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Fibrillar collagen gels were prepared from acid-solubilized rat-tail
collagen I (BD Biosciences), as described previously.^{26 (link),31 (link),32 (link)} Briefly, high-concentration collagen
was diluted to 6 mg/mL with PBS, and then NaOH (1 N) was added to
neutralize the pH; afterward, an equal amount of Dulbecco modified
Eagle medium (DMEM)–Hepes (Gibco) was added to dilute the collagen
to a 3 mg/mL solution. Finally, collagen gels with different physical
cues were generated at different polymerization temperatures (4, 21,
and 37 °C) and corresponding polymerization times of overnight
(15 h), 2 h, and 30 min, respectively.

Xie J., Bao M., Bruekers S.M, & Huck W.T. (2017). Collagen Gels with Different Fibrillar Microarchitectures Elicit Different Cellular Responses. ACS Applied Materials & Interfaces, 9(23), 19630-19637.

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Quantification of N-Myristoyl-D-Asparagine

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N-Myristoyl-d-asparagine levels were quantified in cultures grown for 24 h in 9.5 mL DMEM-HEPES (Gibco). For details, see the Supplemental Material.

Rehm N., Wallenstein A., Keizers M., Homburg S., Magistro G., Chagneau C.V., Klimek H., Revelles O., Helloin E., Putze J., Nougayrède J.P., Schubert S., Oswald E, & Dobrindt U. (2022). Two Polyketides Intertwined in Complex Regulation: Posttranscriptional CsrA-Mediated Control of Colibactin and Yersiniabactin Synthesis in Escherichia coli. mBio, 13(1), e03814-21.

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Bacterial Activation and Isolation Protocol

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The used bacterial strains, plasmids and primers are listed (Supplementary file 2 and 3).
Activating conditions: Following the procedure in (Kenny et al., 1997 (link)), bacterial strains were grown overnight (O/N) in LB medium (Sigma, Israel) at 37°C without shaking, diluted 1:40 into DMEM-HEPES (Gibco, Israel) medium and incubated for 3 hr at 37°C without shaking to exponential phase (O.D.~0.3).
Non-activating conditions: bacteria were plated in LB agar at a concentration below 200 cfu/plate and incubated at 32°C.
For analysis of bacteria isolate directly from colonies, BIG and SMALL colonies were collected according to their size at 17 hr (1000 min) after plating and diluted in 0.9% NaCl to density of ~10⁸ bacteria/ml.

Ronin I., Katsowich N., Rosenshine I, & Balaban N.Q. (2017). A long-term epigenetic memory switch controls bacterial virulence bimodality. eLife, 6, e19599.

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Preparation of Specialized Culture Media

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Preparation of culture media ESM2 and EMS4 was carried out as described [19 (link)]. The basic fibroblast growth factor (bFGF; Gibco, Carlsbad, CA, USA) and medaka embryo extract (MEE) content of ESM2 is higher than ESM4. For ESM4, each 1 L of DMEM/HEPES (pH 7.8; Gibco, New York, NY, USA) contains the following components: FBS (150 mL; Gibco, NSW, Australia), MEE (1 mL; 400 medaka embryos/mL; 2.5 mL for ESM2), fish serum (10 mL; Seabass; serum extraction method as described [16 (link)]), bFGF (20 µL; 0.1 mg/mL; 100 µL for ESM2), L-glutamine, nonessential amino acids, sodium pyruvate, and pen/strep (all 100×; 10 mL; Gibco, New York, NY, USA), 2-mercaptoethanol (4 mL; stock 50 mM; Sigma, Burlington, MA, USA) and sodium selenite (1 mL; stock 2 µM; Sigma). The culture medium was filtered using a filter (0.22 µm) and stored (4 °C) for up to 6 months.

Chen X., Kan Y., Zhong Y., Jawad M., Wei W., Gu K., Gui L, & Li M. (2022). Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro. Biology, 11(7), 1069.

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Quantitative Lipidomic Analysis of E. coli

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E. coli strains were grown for 24 h at 37 °C in 10 ml DMEM-HEPES (Gibco), resuspended in 500 µl Hanks' balanced salt solution (HBSS; Invitrogen) and then crushed with a Precellys instrument (Ozyme). After addition of an internal standard mixture (deuterium-labelled compounds; 400 ng ml⁻¹), cold methanol (MeOH) was added and samples were solid-phase extracted on HLB plates (OASIS HLB 2 mg, 96-well plate; Waters). Lipids were eluted with MeOH, evaporated under N₂, resuspended in MeOH and analysed by HPLC/MS-MS analysis (LC-MS/MS) (MetaToulLipidomics Facility), as described previously [21 (link)].

Auvray F., Perrat A., Arimizu Y., Chagneau C.V., Bossuet-Greif N., Massip C., Brugère H., Nougayrède J.P., Hayashi T., Branchu P., Ogura Y, & Oswald E. (2021). Insights into the acquisition of the pks island and production of colibactin in the Escherichia coli population. Microbial Genomics, 7(5), 000579.

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