Ex cell 293 medium

Manufactured by Merck Group

Ex-Cell 293 medium is a cell culture medium designed for the growth and maintenance of HEK 293 cells. It provides the necessary nutrients and growth factors to support the proliferation and viability of these cells in vitro.

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Lab products found in correlation

Freestyle 293 expression medium, by Thermo Fisher Scientific (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Pei max, by Polysciences (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Expi293 medium, by Thermo Fisher Scientific (1 mentions) Expi293f cells, by Thermo Fisher Scientific (1 mentions) 293t cell, by American Type Culture Collection (1 mentions) Histrap hp column, by Cytiva (1 mentions) Hiprep 26 10 desalting column, by Cytiva (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Trypsinogen, by Merck Group (1 mentions) Enterokinase, by Merck Group (1 mentions) Alexa fluor 488 af488, by Thermo Fisher Scientific (1 mentions) Nickel nta, by Qiagen (1 mentions) Polyethylenimine pei transfection reagent, by Polysciences (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Pei transfection reagent, by Polysciences (1 mentions) Glutamine, by Merck Group (1 mentions) P3000 reagent, by Thermo Fisher Scientific (1 mentions) Puromycin, by Merck Group (1 mentions)

8 protocols using ex cell 293 medium

Cell Culture Protocols for 293T, 293S, and Expi293F

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293T cells (ATCC) were cultured in DMEM medium supplemented with10% fetal bovine serum (FBS). 293S GnTI- cells [27 (link)] and Expi293F cells (Thermo Fisher Scientific) were cultured in suspension [28 (link)] in serum-free Ex-Cell 293 medium (Sigma) and Expi293 medium (Thermo Fisher Scientific), respectively.

Lu C., Song G., Beale K., Yan J., Garst E., Feng J., Lund E., Catteruccia F, & Springer T.A. (2020). Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines. PLoS ONE, 15(1), e0216260.

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Production of SARS-CoV-2 Spike RBD-SD1 Protein

Cited 1 time

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To obtain recombinant His- and Fc-tagged SARS-CoV-2 Spike RBD-SD1 protein, suspension-adapted and serum-free HEK293-S cells were transiently transfected with a pαH expression vector containing the coding sequence of SARS-CoV-2 RBD-SD1 (residues 319–591) upstream of a C-terminal HRV3C protease cleavage site, a monomeric Fc tag and an 8 × His tag (kind gift from Jason McLellan).²² (link) Briefly, cells were seeded in FreeStyle 293 expression medium (Gibco) at a cell density of 3.0 × 10⁶ cells/mL. Cells were transfected by adding 4.5 μg expression vector per mL cells for 5 min, followed by the addition of 9 μg polyethylenimine (Polysciences) per mL cells. After 5 h of incubation at 37 °C, an equal volume of EX-CELL 293 medium (Sigma-Aldrich) was added. After 3 days, 5 g/L d-glucose was added to extend the cell viability and supernatant was harvested 4 days post-transfection. The supernatant was cleared by centrifugation (15 min at 250×g) and filtration. The protein was then purified from the supernatant by immobilized metal ion affinity chromatography (HisTrap HP column, Cytiva), buffer exchanged to PBS by using a HiPrep 26/10 desalting column (Cytiva), and finally filtered over a low protein binding 0.2 μm filter.

Acar D.D., Witkowski W., Wejda M., Wei R., Desmet T., Schepens B., De Cae S., Sedeyn K., Eeckhaut H., Fijalkowska D., Roose K., Vanmarcke S., Poupon A., Jochmans D., Zhang X., Abdelnabi R., Foo C.S., Weynand B., Reiter D., Callewaert N., Remaut H., Neyts J., Saelens X., Gerlo S, & Vandekerckhove L. (2024). Integrating artificial intelligence-based epitope prediction in a SARS-CoV-2 antibody discovery pipeline: caution is warranted. eBioMedicine, 100, 104960.

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Granzyme Purification and Protease Assays

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Human GzmA and GzmB expression plasmids (4 (link)) were transfected into HEK 293T cells by calcium phosphate precipitation. The transfected cells were grown in serum-free ExCell 293 medium (Sigma) for 4 days. Recombinant granzymes were purified from the culture supernatants by immobilized metal affinity chromatography using Nickel-NTA (Qiagen) following the manufacturer’s instructions. Eluted granzymes were treated with enterokinase (0.05 IU/mL supernatant; Sigma) for 16 hours at room temperature. Active Gzms were finally purified on an S column, concentrated, and quality tested as previously described (14 ). GST-tagged HuR, hnRNPC1, and LMNB1 were expressed and purified as described (4 (link)). H1 (NEB M2501S) and caspase 3 (Enzo – ALX-201-059-U025) were purchased. Other serine proteases were NE (Athens Research and Technology, Athens, GA 16-14-051200), PE (Millipore 324682), CATG (Athens Research and Technology 16-14-030107) and trypsinogen (Sigma T1143). Proteins were fluorescently labeled with Alexa Fluor® 488 (AF488) according to the manufacturer’s instructions (Invitrogen A30006).

Thomas M.P., Whangbo J., McCrossan G., Deutsch A., Martinod K., Walch M, & Lieberman J. (2014). Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5390-5397.

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Expression and Purification of hSARM1 Protein

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hSARM1 and mutants (residues 28–724 in PSF-CMV-AMP,
N-terminal His₆-tag, TEV-protease cleavage site, AVI-tag) (Figley et al., 2021 (link)) were expressed in
HEK293S cells (ATCC) and purified to homogeneity using a combination of IMAC
and SEC. The HEK293S cell experiments were performed in accordance with
Griffith University Research Ethics Committee approval 2020/246. The cells
(were grown in 50 % Freestyle 293 Expression Medium (Gibco) and 50 % Ex-Cell
293 Medium (Sigma) supplemented with 3 % L-glutamine in vented flasks at 90
rpm in an 80 % humidified, 8 % carbon dioxide atmosphere at 37°C.
When cells reached a density of 2 × 10⁶ cells/mL,
they were centrifuged at 500 × g for 10 min and
resuspended in 100 % Freestyle 293 Expression Medium to a density of 2.5
×10⁶ cells/mL. After resuspension the cells were
transfected with 3 μg/mL of plasmid DNA using Polyethylenimine
(PEI) transfection reagent (Polysciences) and growth was continued
overnight. On the next day, transfected cells were diluted 1:1 with Ex-Cell
293 Medium and valproic acid was added to a final concentration of 2.2 mM.
Growth was continued for an additional three days. Cells were harvested by
centrifugation at 1,500 × g for 10 min at 4°C
and stored at −80°C until used for purification.

Shi Y., Kerry P.S., Nanson J.D., Bosanac T., Sasaki Y., Krauss R., Saikot F.K., Adams S.E., Mosaiab T., Masic V., Mao X., Rose F., Vasquez E., Furrer M., Cunnea K., Brearley A., Gu W., Luo Z., Brillault L., Landsberg M.J., DiAntonio A., Kobe B., Milbrandt J., Hughes R.O, & Ve T. (2022). Structural basis of SARM1 activation, substrate recognition and inhibition by small molecules. Molecular cell, 82(9), 1643-1659.e10.

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Recombinant CLIMP-63 Luminal Domain Production

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Suspension-adapted HEK293E cells transiently transfected with construct expressing CLIMP-63 Luminal Domain with N-terminal signal recognition peptide and either N- or C-terminal His6-FLAG tag using PEI MAX (Polysciences) in RPMI-1640 (Gibco) supplemented with 0.1% Pluronic-F68. After 1.5 h, cells were diluted into Excell293 medium (Sigma) supplemented with 4 mM glutamine and 3.75 mM valproic acid and agitated for 37 °C. Following a 7-day incubation the cell culture medium was harvested by centrifugation and clarified using a 0.22 µm filter. The conditioned medium was purified by Ni-NTA affinity chromatography via CLIMP63’s His-tag followed by gel-filtration chromatography in 500 mM NaCl, 50 mM HEPES pH 7.5.

Sandoz P.A., Denhardt-Eriksson R.A., Abrami L., Abriata L.A., Spreemann G., Maclachlan C., Ho S., Kunz B., Hess K., Knott G., S. Mesquita F., Hatzimanikatis V, & van der Goot F.G. (2023). Dynamics of CLIMP-63 S-acylation control ER morphology. Nature Communications, 14, 264.

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Transient Protein Expression in HEK293T Cells

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HEK293T cells (ATCC) were grown in 50% Freestyle 293 Expression Medium (Gibco) and 50% Ex-Cell 293 Medium (Sigma) supplemented with 3% L-Glutamine in vented flasks at 90 rpm in an 80% humidified, 8% carbon dioxide atmosphere at 37°C. When cells reached a density of 2 × 10⁶ cells/mL they were centrifuged at 500g for 10 min and resuspended in 100% Freestyle 293 Expression Medium to a density of 2.5 ×10⁶ cells/ml. After resuspension the cells were transfected with 3 μg/mL of plasmid DNA using Polyethylenimine (PEI) transfection reagent (Polysciences) and growth was continued overnight. On the next day, transfected cells were diluted 1:1 with Ex-Cell 293 Medium and valproic acid (VPA) was added to a final concentration of 2.2 mM. Growth was continued for an additional three days. Cells were harvested by centrifugation at 1,500 g for 10 min at 4°C and stored at −80°C until used for purification.

Figley M.D., Gu W., Nanson J.D., Shi Y., Sasaki Y., Cunnea K., Malde A.K., Jia X., Luo Z., Saikot F.K., Mosaiab T., Masic V., Holt S., Hartley-Tassell L., McGuinness H.Y., Manik M.K., Bosanac T., Landsberg M.J., Kerry P.S., Mobli M., Hughes R.O., Milbrandt J., Kobe B., DiAntonio A, & Ve T. (2021). SARM1 is a Metabolic Sensor Activated by an Increased NMN/NAD+ Ratio to Trigger Axon Degeneration. Neuron, 109(7), 1118-1136.e11.

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Establishing Stable Cell Lines for Protein Expression

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PEAK rapid ATCC ® CRL-2828™ cells were seeded in 6-well cell culture plates at a density of 2 × 10⁵ cells per well and incubated in Roswell Park Memorial Institute medium (Sigma-Aldrich) supplemented with 10% of foetal bovine serum (Internegocios) (RPMI-FBS) at 37 °C in 5% CO₂. The medium was removed on the following day and 80–90% confluent cell monolayers were transfected with 2 μg of the recombinant plasmid plus 3.5 μl of Lipofectamine 3000 Reagent (ThermoFisher) and 5 μl of P3000 Reagent (ThermoFisher) in RMPI following the manufacturer's recommendations. At day 1 post-transfection the transfection medium was removed and replaced with RPMI-FBS containing 2 μg /ml of puromycin (Sigma-Aldrich) for selection. The transfected cells were detached from the wells and reseeded in successive passages to T25 or T75 flasks with RPMI-FBS or Ex-Cell 293 medium (Sigma-Aldrich) supplemented with glutamine (Sigma-Aldrich) and 1% FBS. After 5–7 days of culturing, culture supernatants were collected, filtered through a 0.22-nm pore membrane (Millipore, Billerica, MA) and kept at 4 °C.

Villafañe L., Vaulet L.G., Viere F.M., Klepp L.I., Forrellad M.A., Bigi M.M., Romano M.I., Magistrelli G., Fermepin M.R, & Bigi F. (2021). Development and evaluation of a low cost IgG ELISA test based in RBD protein for COVID-19. Journal of Immunological Methods, 500, 113182.

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Purification of CLIMP-63 Luminal Domain

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Suspension-adapted HEK293E cells transiently transfected with construct expressing CLIMP-63 Luminal Domain with N-terminal signal recognition peptide and either N-or C-terminal His6-FLAG tag using PEI MAX (Polysciences) in RPMI-1640 (Gibco) supplemented with 0.1% Pluronic-F68. After 1.5 hours, cells were diluted into Excell293 medium (Sigma) supplemented with 4 mM glutamine and 3.75 mM valproic acid and agitated for 37°C. Following a 7-day incubation the cell culture medium was harvested by centrifugation and clari ed using a 0.22 µm lter. The conditioned medium was puri ed by Ni-NTA a nity chromatography via CLIMP63's His-tag followed by gel-ltration chromatography in 500 mM NaCl, 50 mM HEPES pH 7.5.

Sandoz P.A., Denhardt-Eriksson R., Abrami L., Abriata L.A., Spreemann G., Maclachlan C., S H., Kunz B., Hess K., Knott G., Mesquita F.S., Hatzimanikatis V., & Goot F.G.v.d. (2022). Model-driven understanding of CLIMP-63 structure and S-acylation reveals fine tuning of ER morphology.

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