Ex cell 420 medium

Manufactured by Merck Group

Sourced in United States, United Kingdom, Belgium

The EX-CELL 420 medium is a chemically defined, animal component-free cell culture medium designed for the growth and maintenance of various mammalian cell lines. It provides a consistent and optimized environment for efficient cell culture performance.

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EX-CELL 420 Serum-Free Medium for Insect cells EX-CELL 420

20 protocols using ex cell 420 medium

Mammalian and Insect Cell Culture Protocols

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Example 4

HEK293 cells were cultured at 37° C. under 5% CO2 in Dulbecco's Modified Eagle Medium, supplemented with 10% fetal bovine serum (Wisent), or for Drosophila melanogaster S2 cells, at 25° C. in Ex-Cell 420 Medium (Sigma-Aldrich). Media were supplemented with 100 units/mL penicillin (Thermo-Fisher) and 100 μg/mL streptomycin (Thermo-Fisher). Mammalian cells were transfected with 3.5 μg of plasmid DNA in 35 mm dishes using Lipofectamine 3000 (Thermo-Fisher), or Transit-Insect (Mirus) for Drosophila cells. For extracellular GFP fluorescence reconstitution, HEK293 cells were transfected with constructs expressing GFP1-10 tagged to the transmembrane and extracellular domains of the cell surface molecule Neuroligin-1 and incubated for 24-36 hours. Cells displaying GFP1-10 on the extracellular side of the cell membrane were incubated in culture medium containing 50 μM GFP11 peptide for 3 hours at 37° C. before live imaging. GFP1-10 protein was allowed to accumulate for 24 hours before transfection of GFP11 constructs. For genome editing experiments, 800 ng of CRISPR-Cas9 plasmid DNA were cotransfected with 800 ng of single stranded oligonucleotide repair templates in 12-well plates. After 2-7 days, cells were non-enzymatically dissociated and seeded on glass coverslips and prepared for imaging and electrophysiology experiments.

US11300509B2. Cleavable linkers for protein translation reporting (2022-04-12). 9412-1126 Québec inc. [CA]. Inventors: Brian Chen [CA], El Cheikh Ibrahim Kays [US].

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Recombinant Pfs48/45 Protein Expression

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For the expression of recombinant Pfs48/45 proteins, Drosophila melanogaster S2 cells (ExpreS²ion Biotechnologies, Denmark) were transfected according to manufacturer’s instructions to generate stable polyclonal cell lines. These cells were cultured in EX-CELL420 medium (Sigma-Aldrich) at 25°C. The identity of this cell line has not been authenticated; it was used for recombinant expression of proteins of which the identity was confirmed.

Fabra-García A., Hailemariam S., de Jong R.M., Janssen K., Teelen K., van de Vegte-Bolmer M., van Gemert G.J., Ivanochko D., Semesi A., McLeod B., Vos M.W., de Bruijni M.H., Bolscher J.M., Szabat M., Vogt S., Kraft L., Duncan S., Kamya M.R., Feeney M.E., Jagannathan P., Greenhouse B., Dechering K.J., Sauerwein R.W., King C.R., MacGill R.S., Bousema T., Julien J.P, & Jore M.M. (2023). Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes. Immunity, 56(2), 406-419.e7.

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Recombinant hRET Extracellular Domain Purification

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A codon-optimised human RET^ECD (hRET^ECD) cDNA encoding residues 1-635 followed by a TEV-cleavable Avi and C-tag was cloned into a pExpreS2.1 vector (ExpreS2ion Biotechnologies, Hørsholm, Denmark) with Zeocin resistance. A stable pool of S2 cells, secreting hRET^ECD, was generated by transfecting 25 ml of S2 cells grown in Ex-Cell420 medium (Sigma) with 10 % (v/v) FBS at a density of 5×10⁶ cells/ml using 12.5 μg of DNA and 50 μl of ExpresS²-Insect TR (5×). Stably transfected cells were selected with 2 mg/ml Zeocin with repeated medium exchange. The culture was expanded to 1 litre in a 5L glass-flask and the supernatants collected after 7 days.
For purification, 1 ml of C-tag capture resin (ThermoFisher) was added to a cleared and filtered S2 supernatant and incubated for 18 hrs at 4°C. The resin was pelleted and washed several times with PBS before eluting bound hRET^ECD by competition with PBS containing 200 μg/ml SEPEA peptide. At this point, the affinity and biotinylation tags were removed by digestion with TEV (a 1:10 ratio of TEV protease:RET). The purified hRET^ECD was further purified by size-exclusion using a Superdex200 10/300 with a 50 mM Tris (pH 7.5), 100 mM NaCl buffer.

Adams S.E., Purkiss A.G., Knowles P.P., Nans A., Briggs D.C., Borg A., Earl C.P., Goodman K.M., Nawrotek A., Borg A.J., McIntosh P.B., Houghton F.M., Kjær S, & McDonald N.Q. (2021). A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing. Structure(London, England:1993), 29(7), 694-708.e7.

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Radiolabeled MPEP Binding Assay

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Tritiated 2-methyl-6-(phenylethynyl)pyridine hydrochloride [³H] MPEP was purchased from the American Radiolabeled Chemicals incorporation (ARC), glutamate from Sigma Aldrich, quisqualate and MPEP from Tocris. Sf9 cells adapted in SF-900 II SFM and EX-CELL-420 medium were ordered from Sigma Aldrich, lipofectamine 2000 and DMEM medium from Life Technologies. Detergents were ordered from Anatrace. SNAP-Lumi4-Tb and SNAP Red were obtained from Cisbio Bioassays. Bodipy-FL GTPγS was purchased from ThermoFisher. (R)-5-((3-fluorophenyl) ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl) picolinamide (VU0424465) was synthetized as detailed in the supplementary materials.

Nasrallah C., Rottier K., Marcellin R., Compan V., Font J., Llebaria A., Pin J.P., Banères J.L, & Lebon G. (2018). Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs. Scientific Reports, 8, 4407.

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Baculovirus Production and Purification

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The recombinant baculovirus was amplified in Sf9 cells (Thermo Fisher Scientific, USA) to a density of 2 x 10⁶ cells/mL in ExCell 420 medium (Sigma Aldrich, USA) supplemented with 5% fetal bovine serum (Gibco, USA). Cultures were infected at a multiplicity of infection (MOI) of 0.4. At 48 hours post infection (hpi), cultures were centrifuged at 4500 rpm for 15 minutes. The supernatants were collected and titrated by plaque assay. Viral stocks were stored at 4°C until use.
For protein production, 7 L of Sf9 cell culture at a density of 2 x 10⁶ cells/mL were infected with the baculovirus at a MOI of 3 using a Biostat B plus bioreactor (Sartorius, Germany). The following conditions were maintained during the culture period: temperature at 28°C, pH at 6.2, 50% dissolved oxygen (DO) with an oxygen flow rate of 0.1 vvm via micro sparger and agitation at 150 rpm. At 48 hours post-infection, the cultures were centrifuged at 4500 rpm for 15 minutes and the supernatant was filtered through a 0.22 μm membrane.

Choque-Guevara R., Poma-Acevedo A., Montesinos-Millán R., Rios-Matos D., Gutiérrez-Manchay K., Montalvan-Avalos A., Quiñones-Garcia S., Cauti-Mendoza M.D., Agurto-Arteaga A., Ramirez-Ortiz I., Criollo-Orozco M., Huaccachi-Gonzales E., Romero Y.K., Perez-Martinez N., Isasi-Rivas G., Sernaque-Aguilar Y., Villanueva-Pérez D., Ygnacio F., Vallejos-Sánchez K., Fernández-Sánchez M., Guevara-Sarmiento L.A., Fernández-Díaz M, & Zimic M. (2022). Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models. PLoS ONE, 17(8), e0269823.

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Culturing Lenti-X 293T and Sf9 Cells

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Lenti-X 293T cells were cultured and maintained in Dulbecco’s modified Eagle medium (DMEM) (Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) (GE Healthcare) and 0.2% penicillin-streptomycin solution (Gibco/Invitrogen, Paisley, UK) at 37 °C with 5% CO₂. Spodoptera frugiperda (Sf9) cells (Invitrogen) were maintained in EX-CELL 420 medium (Sigma, Gillingham, UK) supplemented with 2% fetal bovine serum and 1% penicillin/streptomycin solution at 27 °C with shaking.

Alsaadi E.A., Neuman B.W, & Jones I.M. (2020). Identification of a Membrane Binding Peptide in the Envelope Protein of MHV Coronavirus. Viruses, 12(9), 1054.

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Recombinant baculovirus protein expression

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Spodoptera frugiperda Sf9 cells (Millipore) were maintained in Ex-Cell 420 medium (Sigma), and recombinant baculoviruses were generated with the Bac-to-Bac baculovirus expression system as previously described (Izumiya et al., 2009 (link); Kim et al., 2013 (link)). Transfer plasmid, pFAST-BAC1 vector was modified by inserting a Flag tag at the N-terminus, and CHD4, ORF57, p65, and Luciferase cDNAs were cloned into the CpoI (RsrII) site. The cDNA of ADNP, which also include C-terminal His tag was synthesized and cloned into BamHI and PstI restriction enzyme sites by HiFi Gene assembly (NEB). Recombinant baculovirus bacmid DNA was transfected into Sf9 cells by using polyethylenimine (Sigma), and recombinant viruses were subsequently amplified twice. Expression of recombinant proteins was confirmed by immunoblotting with anti-Flag monoclonal antibody (Sigma) or anti-His (BioRad). Largescale cultures of Sf9 cells (50 mL) were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 0.1–1.0, and cells were harvested 48 hrs after infection. Recombinant proteins were purified as described previously (Izumiya et al., 2005 (link)). The purity and amount of protein were measured by SDS-PAGE and Coomassie blue staining, using bovine serum albumin (BSA) as a standard.

Kumar A., Lyu Y., Yanagihashi Y., Chantarasrivong C., Majerciak V., Salemi M., Wang K.H., Inagaki T., Chuang F., Davis R.R., Tepper C.G., Nakano K., Izumiya C., Shimoda M., Nakajima K.I., Merleev A., Zheng Z.M., Campbell M, & Izumiya Y. (2022). KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4. Cell reports, 39(6), 110788.

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Measuring mitochondrial mRNA stability in TcA cells

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TcA cell line was derived from T. castaneum in the late pupal stage⁵⁹. Cells were maintained in Ex-cell 420 medium (Sigma-Aldrich, St. Louis, MO) with 10% Fetal Bovine Serum (FBS, VWR Seradigm, Radnor, PA) at 28 °C. TcA cells were seeded in 12-well plates and treated with TcLRPPRC or malE dsRNA. TcA cells were collected and used to measure mt-mRNA stability at 48 h after treatment following the methods described previously with slight modifications^{60 (link)}. The cells were treated with 1 μg/ml ethidium bromide (EtBr) to stop mitochondrial transcription. At 0, 2, 4, and 6 h after treatment with EtBr, the cells were collected into TRI reagent (Molecular Research Centre Inc., Cincinnati, OH). The mRNA levels were determined as described above.

Jiao Y, & Palli S.R. (2022). Mitochondria dysfunction impairs Tribolium castaneum wing development during metamorphosis. Communications Biology, 5, 1252.

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Cultivation of Insect Cell Lines

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The selected cell lines were available to the labs participating in this study.
High Five cells (Trichoplusia ni) were maintained at 27.5 °C in IPL-41 Insect Medium (Sigma-Aldrich, Bornem, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies, Merelbeke, Belgium).
TcA cells (Tribolium castaneum) were maintained at 27.5 °C in EX-CELL^® 420 medium (Sigma-Aldrich), supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 5 µg/mL human insulin (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies, Merelbeke, Belgium).
S2 cells (D. melanogaster) were maintained at 25 °C in Shields and Sang M3 Insect Medium (Sigma-Aldrich), supplemented with 1 g/L yeast extract (Sigma-Aldrich), 2.5 g/L Bacto™ Peptone (BD Biosciences, San Jose, CA, USA), 10% heat-inactivated FBS (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies).

Santos D., Mingels L., Vogel E., Wang L., Christiaens O., Cappelle K., Wynant N., Gansemans Y., Van Nieuwerburgh F., Smagghe G., Swevers L, & Vanden Broeck J. (2019). Generation of Virus- and dsRNA-Derived siRNAs with Species-Dependent Length in Insects. Viruses, 11(8), 738.

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Culturing Expi293F and Drosophila S2 Cells

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Expi293F cells (Thermo Fisher Scientific) were maintained in suspension in Expi293 expression medium (Thermo Fisher Scientific) at 37 °C, 8% CO₂, in a shaking incubator at 120 rpm.
Drosophila S2 cells^{42 (link)} were cultured at 25 °C using EX-CELL 420 medium (Sigma-Aldrich) with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum at 115 rpm in an Innova 44 shaking incubator.

Ragotte R.J., Pulido D., Lias A.M., Quinkert D., Alanine D.G., Jamwal A., Davies H., Nacer A., Lowe E.D., Grime G.W., Illingworth J.J., Donat R.F., Garman E.F., Bowyer P.W., Higgins M.K, & Draper S.J. (2022). Heterotypic interactions drive antibody synergy against a malaria vaccine candidate. Nature Communications, 13, 933.

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