G418 solution

Manufactured by Merck Group

Sourced in United States

G418 solution is a lab reagent commonly used in cell culture applications. It is a broad-spectrum antibiotic that inhibits protein synthesis, resulting in the death of cells that do not express a resistance gene. The solution is sterile-filtered and ready to use for selection and maintenance of transformed cells.

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G418 disulfate salt solution (8 citations)

9 protocols using g418 solution

Fluorescent Labeling of Ornithine Decarboxylase

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The degron sequence of ornithine decarboxylase (ODC) is recognized directly by proteasomes, leading to the destruction of the involved protein.¹¹, ¹⁷ The retroviral expression vector pQCXIN‐ZsGreen‐cODC, containing the green fluorescent ZsGreen‐labeled ODC degron (Gdeg), was kindly provided by Dr Frank Pajonk (Jonsson Comprehensive Cancer Center, UCLA). LPACs express ZsGreen and could be detected by fluorescent microscopy or flow cytometry.
The plasmid was transfected into platinum retroviral packaging cells using Lipofectamine 2000 (Thermo Fisher Scientific). The retrovirus collected from the supernatant was used for infection. Stable transfectants were selected with G418 solution (Sigma‐Aldrich) and maintained in 0.1 mg/ml G418 solution.

Ichihara M., Takahashi H., Nishida N., Ivan C., Okuzaki D., Yokoyama Y., Ohtsuka M., Miyoshi N., Uemura M., Tanaka S., Calin G.A., Mori M., Doki Y., Eguchi H, & Yamamoto H. (2022). Long noncoding RNA 01534 maintains cancer stemness by downregulating endoplasmic reticulum stress response in colorectal cancer. Annals of Gastroenterological Surgery, 7(3), 458-470.

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Cell Culture Protocols for Cancer and Endothelial Cells

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LNCaP (ATCC, CRL—1740), Du-145 (ATCC, CRL—HTB—81), PC3 (ATCC, CRL—1435), C4-2 (ATCC, CRL—3314), HEK293E (ATCC, CRL—10852) and Jurkat cells (ATCC, TIB-152) were cultured in RPMI medium 1640 (Gibco, Waltham, Massachusetts, USA #61870-010) supplemented with 10% foetal bovine serum (FBS) (Gibco, #10500-064) and 1% penicillin–streptomycin (Gibco, #15140-122). Human dermal microvascular endothelial cells (HMEC-1) stably overexpressing human FcRn (HMEC-1-FcRn) [18 (link)] were cultivated in complete medium consisting of MCDB131 (Life Technologies, Waltham, Massachusetts, USA #10372-019), Recombinant human EGF (PeproTech, Cranbury, New Jersey, USA #AF-100-15), Hydrocortisone (Sigma-Aldrich, St. Louis, Missouri, USA #H0888), G418 solution (Sigma-Aldrich, #G418-RO), Puromycin (Life Technologies, #A11138-03), 10% FBS, and 2 mM l-glutamine (Lonza, Basel, Switzerland #BE17-605E). Cells were cultivated following ATCC protocols, and mycoplasma tested when new stock were thawed. Cell lines were routinely authenticated by short tandem profiling.

Glud E.N., Rasmussen M., Zhang Y., Mandrup O.A., Salachan P.V., Borre M., Sørensen K.D, & Howard K.A. (2022). Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design. British Journal of Cancer, 127(12), 2186-2197.

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hESC Expansion and Passaging Protocol

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H1-hESC were obtained from Yale Stem Cell Center and used up to passage 90. H9-Fucci-hESC were obtained from Dr. Ludovic Vallier’s lab at Cambridge Stem Cell Institute and used up to passage 90 (Pauklin and Vallier, 2013 (link)). All hESC were cultured in mTeSR™ medium (STEMCELL Technologies cat# 85850) on Matrigel-coated plates (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 5 mL, Corning cat# 356230) as described in Fu et al. (2011) (link). Importantly, 50 μg/ml G-418 solution (Sigma cat# 4727878001) was added to the culture medium for H9-Fucci-hESC to eliminate non-transduced cells. For passage, 160 μl/ml Dispase (STEMCELL Technologies cat# 07913) was added to cell culture. Cells were incubated with Dispase at 37°C for 45min. Afterward, cells were transferred to Falcon tubes containing DMEM:F12 medium (GIBCO cat# 11320–033) for washing. 12ml of DMEM:F12 medium was used per 1ml of cell culture medium. Let tubes stand at room temperature for 5–10min or until all cell pellets settle to the bottom of the tubes. Remove supernatant and cells were resuspended in mTeSR™ medium, dispersed into small clumps, and re-plated onto Matrigel-coated plates.

Qiu J., Nordling S., Vasavada H.H., Butcher E.C, & Hirschi K.K. (2020). Retinoic Acid Promotes Endothelial Cell Cycle Early G1 State to Enable Human Hemogenic Endothelial Cell Specification. Cell reports, 33(9), 108465.

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Retroviral Transduction of Degron-Labeled ODC

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The degron sequence of ornithine decarboxylase (ODC) is recognized directly by proteasomes, which leads to the destruction of the involved protein. The retroviral expression vector pQCXIN-ZsGreen-cODC, containing green fluorescence ZsGreen-labeled degron ODC (Gdeg) was kindly provided by Dr Frank Pajonk (Jonsson Comprehensive Cancer Center, UCLA, CA, USA). The plasmid was transfected into Platinum retroviral packaging cells using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). The plasmid and Lipofectamine^® 2000 were diluted with Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Inc.) for 5 min at room temperature, separately. The diluted Lipofectamine^® 2000 was subsequently mixed with the diluted plasmid and incubated for 15 min at room temperature. The mixture was then added to cells immediately at room temperature, after which the cells were incubated at 37°C. And the retrovirus collected from the supernatant was used for Panc-1 cell infection as previously described (32 (link)-34 (link)). Stable transfectants were selected with G418 solution (Sigma-Aldrich; Merck KGaA) and maintained in 0.1 mg/ml G418 solution.

Wang J., Yokoyama Y., Hirose H., Shimomura Y., Bonkobara S., Itakura H., Kouda S., Morimoto Y., Minami K., Takahashi H., Shibata S., Kobayashi S., Uemura M., Tanaka S., Wu X., Tanaka S., Mori M, & Yamamoto H. (2022). Functional assessment of miR-1291 in colon cancer cells. International Journal of Oncology, 60(2), 13.

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hESC Expansion and Passaging Protocol

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Generating Dox-Inducible Myoblasts from MEFs

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MEFs were isolated from Pax7-nGFP mice and cultured in “MEF medium” containing high-glucose Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, 41966029) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, 10270106), 1% GlutaMAX (Thermo Fisher Scientific, 35050061), 1% nonessential amino acids (Thermo Fisher Scientific, 11140050), 1% penicillin-streptomycin (Pen-Strep; Thermo Fisher Scientific, 15140122), and 0.1% 2-mercaptoethanol (Thermo Fisher Scientific, 21985023). To generate dox-inducible Rep-MEFs, cells were passaged and, once confluent, transduced with LV-EF1α-rtTA3/PGK-Neomycin plus LV-tetO-MyoD/PGK-puromycin at 1:1 ratio and supplemented with polybrene (6 μg/ml; Sigma-Aldrich, TR-1003-G). Transduced MEFs were expanded and selected by sequential antibiotic treatment with MEF medium containing either G418 solution (1 mg/ml; Sigma-Aldrich, 4727878001) or puromycin (1 μg/ml; Thermo Fisher Scientific, A1113803) for a total of 4 days. To establish a homogeneous population of fibroblasts from Rep-MEF cultures, we FACS-purified these cultures with an antibody recognizing the fibroblast-specific surface marker CD90.2 (Thy 1.2) (Thermo Fisher Scientific, 48-0902-80). Cells were FACS-purified or analyzed using an SH800S FACS-Sorter (Sony Biotechnology Inc.).

Kim I., Ghosh A., Bundschuh N., Hinte L., Petrosyan E., von Meyenn F, & Bar-Nur O. (2022). Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells. Science Advances, 8(14), eabj4928.

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HepG2 and bEnd.3 Cell Culture

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HepG2 and bEnd.3 cell lines purchased from ATCC were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, HyClone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, MA, USA) at 37 ℃ and 5% CO₂. HepG2 cell lines constantly expressing the CrebH gene were generated by transfection with pcDNA3-Flag-CrebH [21 (link)] using the Lipofectamine LTX plus reagent system (Invitrogen, CA, USA) and selected by adding G418 solution (Sigma-Aldrich).

Lee S.H., Moon S.J., Woo S.H., Ahn G., Kim W.K., Lee C.H, & Hwang J.H. (2023). CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA. Cell & Bioscience, 13, 116.

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Lentiviral Transduction of Mesenchymal Stem Cells

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Frozen viral supernatants were thawed and diluted 1:1 in serum-free QBSF-60 media (Quality Biological, Gaithersburg, MD, United States) and 8 μg/ml of protamine sulfate (Calbiochem, San Diego, CA, United States). BM-MSC, UCT-MSC, UCT-EPC, and AF-MSC were transduced at a very low multiplicity of infection (MOI) (approximately, 1 vector to 300 cells, infectious viruses per cell; MOI 0.0032) to ensure single integration events. After overnight incubation at 37°C, cells were cultured in their respective growth medium. Twenty-four hours later, cells underwent antibiotic selection using 350 μg/ml G418 solution (Sigma-Aldrich, St. Louis, MO) for UCT-EPC, or 500 μg/ml G418 disulfate salt solution for BM-MSC, UCT-MSC, and AF-MSC for 5 days, replacing the selection media after 3 days. After antibiotic selection, the remaining transduced cells expressed the neomycin resistance gene (neomycin phosphotransferase II) and the HSQ transgene. Upon confluence, transduced cells were used for supernatant collection, qPCR, and immunofluorescence imaging.

Stem C., Rodman C., Ramamurthy R.M., George S., Meares D., Farland A., Atala A., Doering C.B., Spencer H.T., Porada C.D, & Almeida-Porada G. (2021). Investigating Optimal Autologous Cellular Platforms for Prenatal or Perinatal Factor VIII Delivery to Treat Hemophilia A. Frontiers in Cell and Developmental Biology, 9, 678117.

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Recombinant Decorin Production in CHO Cells

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For proteoglycan production a pCMV expression vector (Thermo Fisher Scientific, Waltham, MA, USA) construct containing the human decorin cDNA sequence was created (Szilák Laboratories, Bioinformatics & Molecule-design Ltd. Szeged, Hungary). Next, CHO-S (Chinese hamster ovary) cells (Gibco/Thermo Fisher) were transfected by the expression vector using electroporation by Neon™ transfection system, following manufacturer's recommendations (Invitrogen/Thermo Fisher). Successfully transfected cells were selected by adding 500 μg/ml of G418 solution to cell culture medium (Sigma) and human recombinant decorin production was conducted following manufacturer's recommendations (Gibco/Thermo Fisher). The recombinant proteoglycan was isolated as previously described [28] (link). After chromatographic purification the samples were dialyzed against distilled water and the proteoglycan content was checked by dimethyl methylene blue (DMB, Sigma-Aldrich, St. Luis, MO, USA) staining. Finally, salts were added to the samples to convert the distilled water to 1x phosphate buffered saline (PBS), and the final decorin concentration was adjusted to 1000 μg/ml. These stock solutions were stored at -20˚C until use.

Horváth Z., Reszegi A., Szilák L., Dankó T., Kovalszky I, & Baghy K. (2019). Tumor-specific inhibitory action of decorin on different hepatoma cell lines. Cellular signalling, 62.

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