Cd293

Manufactured by Thermo Fisher Scientific

Sourced in Italy

CD293 is a serum-free, animal component-free medium designed for the cultivation of Chinese Hamster Ovary (CHO) cells and other mammalian cell lines. It supports cell growth and protein production, providing a reliable and consistent performance for bioproduction applications.

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CD293 medium (10 citations)

16 protocols using cd293

Recombinant sPTPRG-Fc Fusion Protein

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A recombinant sPTPRG derivative consisting of the extracellular domain without transmembrane segment and cytoplasmic tail, was expressed in HEK 293F cells. To obtain the PTPx-FcIgG3 construct, the cDNA fragment corresponding to extracellular domains of PTPRG 1 through 736 was cloned into pCEP4 plasmid carrying a mouse IgG3 domain between KpnI and BamHI and was inserted in frame between Bcl I (inside IgG3 domain) and Kpl I. Transfected HEK 293F cells were cultured in RPMI 1640 containing 10% heat-inactivated Fetal Calf Serum (FCS), 4mM glutamine, and 0,5 mg/mL of hygromycin (Invitrogen Milan, Italy) as selective agent or cultured in serum free medium CD293 (Gibco, Milan, Italy) with 50 μM β-mercaptoethanol and 0,5 mg/mL of hygromycin (Invitrogen, Milan, Italy). All the cultured cells were grown at 37°C, in 5% CO₂. The recombinant ECD-PTPRG-Fc fusion protein was purified from serum free medium cultured cell by affinity chromatography using protein-G Sepharose according to the manufactory protocol of HiTrap Protein G HP, 1 ml (GE Healthcare, Milan, Italy). The yield was around 0,2 mg/ml for >80% of purity.

Moratti E., Vezzalini M., Tomasello L., Giavarina D, & Sorio C. (2015). Identification of Protein Tyrosine Phosphatase Receptor Gamma Extracellular Domain (sPTPRG) as a Natural Soluble Protein in Plasma. PLoS ONE, 10(3), e0119110.

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Measuring Human OR5AN1 Activity

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In vitro activity of the human OR5AN1 was measured using the Dual-Glo Luciferase Assay System (Promega). Hana3A cells were cotransfected with the OR, a short form of receptor transporter protein 1 (RTP1S), the type 2 muscarinic acetylcholine receptor (M3-R), Renilla luciferase driven by an SV40 promoter, and firefly luciferase driven by a cyclic adenosine 5′-monophosphate response element. Eighteen to 24 hours after transfection, ORs were treated with medium or serial dilutions of odorants spanning 1 nM to 1 mM in triplicate. Odors were first diluted to 1 M stocks in dimethyl sulfoxide and then diluted from stocks to the appropriate concentration in CD293 (Gibco). Four hours after odorant stimulation, luciferase activity was measured using the Synergy 2 (BioTek). Normalized luciferase activity was calculated by dividing firefly luciferase values by Renilla luciferase values for each well. Results represent mean response (for three wells) ± SEM. Responses were fit to a three-parameter sigmoidal curve.

Saraiva L.R., Riveros-McKay F., Mezzavilla M., Abou-Moussa E.H., Arayata C.J., Makhlouf M., Trimmer C., Ibarra-Soria X., Khan M., Van Gerven L., Jorissen M., Gibbs M., O’Flynn C., McGrane S., Mombaerts P., Marioni J.C., Mainland J.D, & Logan D.W. (2019). A transcriptomic atlas of mammalian olfactory mucosae reveals an evolutionary influence on food odor detection in humans. Science Advances, 5(7), eaax0396.

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Dual-Glo Luciferase Assay for Odorant Receptor

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In the large-scale screening, the Dual-Glo Luciferase Assay System (Promega) was used to measure receptor responses as previously described^{31 (link)}. Hana3A cells were plated on 96-well plates. Approximately 18–24 h after plating, cells were transfected with 5 ng/well of plasmids encoding OR, 2.5 ng/well of M3-R, 5 ng/well of RTP1S, 10 ng/well of CRE-luciferase and 5 ng/well of pRL-SV40. Furthermore, 18–24 h later, cells were stimulated by incubation with an odorant diluted in CD293 (Gibco) at 37 °C and 5% CO₂ to allow for CRE-luciferase expression. Four hours after stimulation, luminescence was measured a Polarstar Optima plate reader (BMG). All luminescence values were divided by Renilla luciferase activity to control for transfection efficiency in a given well. Each comparison was performed in triplicate. Statistical significance was assessed by a two-sided t-test comparing the wells stimulated with odor with the three wells stimulated with medium alone.

Kida H., Fukutani Y., Mainland J.D., de March C.A., Vihani A., Li Y.R., Chi Q., Toyama A., Liu L., Kameda M., Yohda M, & Matsunami H. (2018). Vapor detection and discrimination with a panel of odorant receptors. Nature Communications, 9, 4556.

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PAPP-A2 Overexpression and Secretion in 293T Cells

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Human embryonic kidney 293T cells (293tsA1609neo, ATCC CRL‐3216) (Pear et al, 1993) were maintained in high‐glucose DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, and gentamicin (Invitrogen). The cells are routinely tested for mycoplasma contamination. For transient transfection, 6.0 × 10⁶ cells were plated onto 10‐cm dishes and transfected 18 h later by calcium phosphate coprecipitation using 10 μg plasmid DNA (Overgaard et al, 2000). In addition to plasmid constructs encoding PAPP‐A2 or mutated variants, constructs encoding human IGFBP‐3 (Laursen & Oxvig, 2005), human IGFBP‐4 (Boldt et al, 2001), or human IGFBP‐5 (Overgaard et al, 2001) were used. Culture supernatants were harvested 48 h post‐transfection and cleared by centrifugation, or the cells were further cultured in serum‐free medium (CD293, Invitrogen) to facilitate purification of the secreted proteins. Concentrations of WT PAPP‐A2 in the media were determined by a two‐site ELISA (Laursen et al, 2007).

Dauber A., Muñoz‐Calvo M.T., Barrios V., Domené H.M., Kloverpris S., Serra‐Juhé C., Desikan V., Pozo J., Muzumdar R., Martos‐Moreno G.Á., Hawkins F., Jasper H.G., Conover C.A., Frystyk J., Yakar S., Hwa V., Chowen J.A., Oxvig C., Rosenfeld R.G., Pérez‐Jurado L.A, & Argente J. (2016). Mutations in pregnancy‐associated plasma protein A2 cause short stature due to low IGF‐I availability. EMBO Molecular Medicine, 8(4), 363-374.

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Production and Characterization of PI-Tf Fusion Protein

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Example 1

PI-Tf Recombinant Fusion Protein Expression and Characterization

Preproinsulin sequence (NM_000207) (SEQ ID NO: 1) fused in frame with Tf sequence (NM_001063) (SEQ ID NO: 2) was engineered into pcDNA3.1 (+) expression vector (Invitrogen, CA) by molecular cloning methods (FIG. 1). Plasmids containing preproinsulin-Tf fusion gene were transiently transfected to HEK 293 cells through polyethylenimine-mediated DNA transfection. Conditioned serum-free media were collected and concentrated by labscale tangential flow filtration system (Millipore, MA), and then ultrafiltered (CENTRICON®, Millipore, MA). PI-Tf fusion protein was characterized and quantified by Western blot using both anti-Tf (Sigma, MO) and anti-(pro)insulin antibodies (Abeam, MA). Anti-Tf and anti-(pro)insulin Western blots demonstrated the presence of a major band with molecular weight ˜89 kD, which indicated that PI-Tf fusion protein was successfully expressed and secreted into media. A leucine-glutamate dipeptide sequence was introduced between proinsulin and Tf due to the Xhol restriction enzyme cutting site. The Tf shown on Lane 3 of FIG. 2 came from the original serum-free cell culture medium, CD 293 (Invitrogen), instead of production from transfected HEK293 cells. The dipeptide linker remained stable during production process.

US10513563B2. Method for uses of proinsulin transferrin fusion proteins as prodrugs (2019-12-24). None [US], None [US], None [US]. Inventors: Wei-Chiang Shen [US], Yan Wang [US], Jennica Krankel [US].

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Generation of Whole IgG Antibodies

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To convert the selected Fabs into whole IgG format, the VH and VK sequences were amplified by PCR and combined with the leader sequences of IgG heavy and light chains, respectively, by overlap extension PCR using Pfu DNA Polymerase (Thermo Scientific). The VH and VK with leader sequences were sequentially cloned into the EcoRI-ApaI and HindIII-BsiWI sites, respectively, in the antibody expression cassette (pdCMV-dhfrC) containing the human constant region of γ1 heavy chain (Cγ1) and Cκ gene³⁶. For transient IgG expression, the resulting expression plasmid was introduced into HEK293T cells using Lipofectamine (Invitrogen) according to the manufacturer’s instructions, and the transfected cells were cultured in protein-free medium (CD293, Invitrogen). The IgG was purified from the culture supernatant by affinity chromatography on protein A (Millipore), and its expression and purity were analyzed by western blotting and SDS-PAGE, respectively. The protein concentration was determined by UV spectrophotometry (NanoDrop; Thermo Fisher Scientific).

Guk K., Kim H., Lee M., Choi Y.A., Hwang S.G., Han G., Kim H.N., Kim H., Park H., Yong D., Kang T., Lim E.K, & Jung J. (2020). Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus. Nature Communications, 11, 3418.

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High-Throughput Screening of GPCR Activation

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Hana3A cells were plated onto 96-well plates (Greiner bio-one) with 50 µl growth medium (DMEM medium with 10% BCS and 5% penicillin–streptomycin solution with puromycin) and cultured at 37 °C with 5% CO₂. After 18–24 h, cells in each well were cotransfected with 50 ng receptor plasmid, 10 ng CRE-Luc, 10 ng pRL-SV40, and 10 ng mRTPs using Lipofectamine 2000 (Invitrogen), and incubated for 18 h. Medium was then replaced by 25 µl CD293 (Invitrogen) containing 1% glutamine with or without test compounds for 4 h. The chemiluminescence of firefly luciferase and renilla luciferase were measured with a Biotek Microplate reader using the Dual-Glo Luciferase Assay System (Promega).

Guo L., Dai W., Xu Z., Liang Q., Miller E.T., Li S., Gao X., Baldwin M.W., Chai R, & Li Q. (2022). Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors. Molecular Biology and Evolution, 39(3), msac006.

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Isolation and Culture of Prostate Cancer Cells

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Trizol LS Reagent kit (Invitrogen, Carlsbad, CA, USA), TaKaRa PrimeScript RT Reagent Kit (Takara Biotech, Beijing, China), Fluorescein isothiocyanate isomer conjugated with PSA (FITC-PSA) ( Sigma, Saint Louis, MO, USA), PI (Sigma, Saint Louis, MO, USA), Roswell Park Memorial Institute Medium (RPMI Medium) (Gibco, Grand Island, NY, USA), Fetal bovine serum (FBS) (Pan, Aidenbach, Germany), TransIntro^TM PL Transfection Reagent (Tran, Beijing, China), CD293 (Invitrogen, Carlsbad, CA, USA), Dual-Glo Luciferase Assays System (Promega, Madison, WI, USA), non-targeting siRNA oligonucleotides (siRNA NC) (Ribobio, Guangzhou, China), silencing siRNA targeted against the OR2C1 gene (siRNA OR2C1) (Ribobio, Guangzhou, China).Unless stated, all chemicals used were purchased from Sigma (Sigma, Saint Louis, MO, USA). BTS (37 g glucose, 3 g trisodium citrate, 1.25 g Na₂-EDTA, 1.25 g NaHCO₃, 0.75 g KCl, 0.6 g/L penicillin G sodium, and 1.0 g/L dihydrostreptomycin; diluted to 1 L).

Yuan X., Wang Y., Ali M.A., Qin Z., Guo Z., Zhang Y., Zhang M., Zhou G., Yang J., Chen L., Shen L., Zhu L, & Zeng C. (2023). Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin. International Journal of Molecular Sciences, 24(2), 1664.

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Dual-Glo Luciferase Assay for Olfactory Receptors

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The Dual-Glo luciferase assay system (Promega) was used for the luciferase assay (15 (link)). HEK293T cells were plated on poly-d-lysine–coated 96-well plates (Thermo Fisher Scientific). Plasmid DNAs of ORs and RTP1S were transfected using Lipofectamine 2000 (Invitrogen). In addition, two luciferase constructs were used, including a firefly luciferase gene driven by a cAMP-response element (CRE-Luc) and a Renilla luciferase gene driven by a constitutively active SV40 promoter (pRL-SV40) that was used as an internal control for cell viability and transfection efficiency. For each 96-well plate, 10 ng of CRE-Luc, 5 ng of pRL-SV40, 5 ng of OR, and 5 ng of RTP1S were transfected. Twenty four hours post-transfection, the medium was replaced with 25 μl of odorant solution diluted in CD293 (Invitrogen) and incubated for 3.5 h at 37 °C in 5% CO₂. We followed the manufacturer's protocols for measuring firefly luciferase (Luc) and Renilla luciferase (RL) activities. Luminescence was measured using a GloMax-Multidetection System (Promega). The relative response of ORs was calculated as (Luc/RL − Luc/RL_min)/(Luc/RL_max − Luc/RL_min), where Luc/RLN represents the mean value from the replicate wells of a certain sample, and Luc/RLmin represents the mean value from the minimal response in the experiment.

Fukutani Y., Tamaki R., Inoue R., Koshizawa T., Sakashita S., Ikegami K., Ohsawa I., Matsunami H, & Yohda M. (2019). The N-terminal region of RTP1S plays important roles in dimer formation and odorant receptor-trafficking. The Journal of Biological Chemistry, 294(40), 14661-14673.

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Purification of PAPP-A Protein Variants

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Human embryonic kidney 293T cells (293tsA1609neo) were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, nonessential amino acids, and gentamicin (Invitrogen). Cells were plated onto 6-cm tissue culture dishes and transiently transfected 18 h later by calcium phosphate co-precipitation using 10 μg of plasmid DNA. The plasmids encoded the following: Wild-type human PAPP-A [45 (link)], and mutants E483Q [45 (link)] and D1484A [26 (link)], wild-type murine PAPP-A [46 (link)], human c-Myc-His tagged IGFBP-4 [47 (link)], or a c-Myc-His tagged version of plasmid PA(1133–1547) [22 (link)], encoding a C-terminal fragment of human PAPP-A, starting at SCR module 1, PAPP-A(1133-1547). After 48 h, the culture media were harvested and cleared by centrifugation, or the cells were further cultured in serum free medium, CD293 (Invitrogen), to facilitate purification. Tagged proteins were purified by nickel affinity chromatography using Chelating-Sepharose Fast Flow beads (1 ml) (Amersham Biosciences). The column was washed with 1 M NaCl, 50 mM sodium phosphate, pH 5.5, and bound protein was eluted with phosphate-buffered saline containing 20 mM EDTA.

Mikkelsen J.H., Resch Z.T., Kalra B., Savjani G., Kumar A., Conover C.A, & Oxvig C. (2014). Indirect targeting of IGF receptor signaling in vivo by substrate-selective inhibition of PAPP-A proteolytic activity. Oncotarget, 5(4), 1014-1025.

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