Optipro serum free medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

OptiPRO serum-free medium is a cell culture medium designed for the growth and maintenance of a variety of mammalian cell lines. It is a liquid, serum-free formulation optimized to support cell proliferation and viability without the use of animal-derived components.

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Lab products found in correlation

21 protocols using optipro serum free medium

Hydrogen Sulfide Inhibition and Copper Counteraction in hFGE Production

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Example 6

Hydrogen sulfide (H₂S) is a putative side product of the FGE catalytic cycle. Sulfide quinone reductase (SQR) is a mitochondrial enzyme that oxides and detoxifies H₂S. Experiments were performed to determine whether H2S accumulation over the course of the fed batch inhibited human FGE activity, and if Cu²⁺ counteracted this effect.

Cells were plated. DNA was mixed with Optipro serum free medium (Life Technologies). FreeStyle Max transfection reagent (Life Technologies) and Optipro serum free medium were mixed. DNA and reagents were combined for 10-20 min at room temperature. The resulting complexes were added the cells.

A Western blotting was performed on lysates from cells treated with Cu²⁺ or not treated with Cu²⁺, using antibodies against SQR to determine whether Cu²⁺ increased the levels of SQR. No significant difference in the levels of SQR was observed. Experiments were also performed to test whether overexpression of SQR in Expi cells could be a substitute for Cu²⁺ in increasing conversion. The results indicated that there was no significant difference in conversion in SQR overexpressing cells compared to the control. SQR overexpression was confirmed in a CHO transient transfection. Thus, SQR is not likely involved in maintaining high conversion in the stationary phase.

US10683527B2. Activated formylglycine-generating enzymes and methods of producing and using the same (2020-06-16). R.P. Scherer Technologies, LLC [US]. Inventors: David Rabuka [US], Gregory W. deHart [US], Patrick Holder [US], Jeanne Baker [US].

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Optimizing Protein Production with H2S Regulation

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Example 6

Hydrogen sulfide (H2S) is a putative side product of the FGE catalytic cycle. Sulfide quinone reductase (SQR) is a mitochondrial enzyme that oxides and detoxifies H2S. Experiments were performed to determine whether H2S accumulation over the course of the fed batch inhibited human FGE activity, and if Cu²⁺ counteracted this effect.

US11655492B2. Activated formylglycine-generating enzymes and methods of producing and using the same (2023-05-23). R.P. Scherer Technologies, LLC [US]. Inventors: David Rabuka [US], Gregory W. deHart [US], Patrick Holder [US], Jeanne Baker [US].

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Investigating H2S Accumulation and Cu2+ Effects on FGE Activity

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Example 6

Hydrogen sulfide (H₂S) is a putative side product of the FGE catalytic cycle. Sulfide quinone reductase (SQR) is a mitochondrial enzyme that oxides and detoxifies H₂S. Experiments were performed to determine whether H₂S accumulation over the course of the fed batch inhibited human FGE activity, and if Cu²⁺counteracted this effect.

A Western blotting was performed on lysates from cells treated with Cu²⁺or not treated with Cu²⁺, using antibodies against SQR to determine whether Cu²⁺increased the levels of SQR. No significant difference in the levels of SQR was observed. Experiments were also performed to test whether overexpression of SQR in Expi cells could be a substitute for Cu²⁺in increasing conversion. The results indicated that there was no significant difference in conversion in SQR overexpressing cells compared to the control. SQR overexpression was confirmed in a CHO transient transfection. Thus, SQR is not likely involved in maintaining high conversion in the stationary phase.

US11053529B2. Activated formylglycine-generating enzymes and methods of producing and using the same (2021-07-06). R.P. Scherer Technologies, LLC [US]. Inventors: David Rabuka [US], Gregory W. deHart [US], Patrick Holder [US], Jeanne Baker [US].

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Recombinant HCMV gO Protein Production

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The open reading frame of gO derived from HCMV strain TB40-BAC4 [59 (link)] lacking the N-terminal signal peptide (aa 1–28) was cloned into the pFUSE-mIgG2BFc2 vector (Invivogen). 293 cells were transfected with pFUSE-gO-mIgG2BFc2 using polyethylenimine. Three hours after transfection medium was exchanged for OptiPRO serum-free medium (LifeTechnologies) and supernatants were harvested 96 hours after transfection. Total supernatant proteins were precipitated with ethanol/125 mM NaCl to control expression by Western blot analysis. Fc proteins were purified from supernatants by precipitation with protein A sepharose (GE Healthcare) followed by elution using ImmunoPure IgG elution buffer, pH 2.8 (Thermo Scientific). Fc protein amounts were determined by a capture ELISA on NeutrAvidin coated plates (Thermo Scientific) using a biotinylated goat anti-mouse IgG (Fc-specific) antibody (Sigma) to capture Fc proteins and a peroxidase-conjugated goat anti-mouse IgG (Fc-specific) antibody (Sigma) and TMB substrate (BD Biosciences) to detect Fc proteins.

Wu Y., Prager A., Boos S., Resch M., Brizic I., Mach M., Wildner S., Scrivano L, & Adler B. (2017). Human cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry. PLoS Pathogens, 13(4), e1006281.

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Purification and Inactivation of Respiratory Syncytial Virus

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RSV strains A2 (Lewis et al., 1961 ) and Line 19 (Herlocher et al., 1999 (link)) were grown in Vero cells for 6–8 days at an input multiplicity of infection (MOI) of 0.01 PFU/cell using OptiPro serum free medium (Life Technologies, Grand Island, NY) supplemented with 4 mM L-glutamine (Life Technologies). Procedures for isolating RSV from the cells were as described in Le Nouën et al. (Le Nouen et al., 2009 (link)) unless otherwise stated. Cells were harvested by scraping, vortexed to liberate surface-associated virus and clarified by centrifugation. The virus was purified by centrifugation through discontinuous sucrose gradients in a Beckman SW32Ti rotor at 121000 x g for 1.5 hours to remove cytokines and other cell derived macromolecules. Virus was harvested from the interface between the sucrose layers, diluted in RPMI 1640 (Life Technologies) supplemented with 2 mM L-glutamine and pelleted at 8,000 x g for 2 hours in a Sorvall SS-34 rotor to remove the sucrose. Pellets were snap frozen in RPMI 1640 and stored at −80°C until use. Viral titer was determined by plaque assay on Vero cells. RSV was UV-inactivated using a dose of 2400 mJ/cm² with a UVC 500 crosslinker (Hoefer Instruments, Holliston, MA).

Hillyer P., Mane V.P., Chen A., dos Santos M.B., Schramm L.M., Shepard R.E., Luongo C., Le Nouën C., Huang L., Yan L., Buchholz U.J., Jubin R.G., Collins P.L, & Rabin R.L. (2017). Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells. Virology, 504, 63-72.

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Propagation of HCoV OC43 in Vero Cells

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Vero (embryonic African green monkey kidney) cells were purchased from ATCC. The cells were cultivated in OptiPro serum-free medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 4 mM L-glutamine (Sigma-Aldrich) in an incubator at 37°C (CO₂: 5%, relative humidity: >95%). HCoV OC43 was obtained from ATCC and propagated in the same medium. The stocks were frozen at −80°C, and virus titer was determined by TCID₅₀ assay.

Graf C., Bernkop-Schnürch A., Egyed A., Koller C., Prieschl-Grassauer E, & Morokutti-Kurz M. (2018). Development of a nasal spray containing xylometazoline hydrochloride and iota-carrageenan for the symptomatic relief of nasal congestion caused by rhinitis and sinusitis. International Journal of General Medicine, 11, 275-283.

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Genome-wide RNAi Screen for SARS-CoV-2 Host Factors

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Screening was performed using the Human Druggable ON-TARGETplus siRNA library, targeting 2,732 annotated genes (#G-104605, Dharmacon). A549 cells were seeded in 384-well plates (4 × 10³ cells/well) in medium without antibiotics and transfected with 10 nM of pooled siRNAs, using 0.2% DharmaFECT1 transfection reagent (Dharmacon). Non-targeting siRNA siScr, siNXF1 and siCSE1L, were used as controls (Dharmacon). At 48 hours post-transfection, media was removed and cells were infected with the rPR8-GFP strain at an MOI of 5 for 1 hour at room temperature (RT). Viral inoculum was removed and Opti-PRO serum-free medium was added (Gibco). At 9 hours post-infection, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) containing 2.5 μg/mL Hoechst 33342 for 30 minutes at RT. Cells were imaged using a high-throughput confocal fluorescence imaging system (Evotec Technologies High-Throughput Cell Analyzer Opera, Perkin Elmer) and analyzed with a previously described in-house image mining (IM) platform51 (link). The screen was conducted in duplicate. The scatter plot was generated using TIBCO Spotfire 4.5.0 (TIBCO Software Inc.). The Z factor was defined by the means and standard deviations of the percentage of cells infected in the positive and negative controls, and is used to assess the quality of a screening assay52 (link).

Lee J., Kim J., Son K., d’Alexandry d’Orengiani A.L, & Min J.Y. (2017). Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry. Scientific Reports, 7, 43893.

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Labeling Neural Stem Cells In Vivo

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For specific expression in NSCs in vivo, the mouse Gfap promoter^{29 (link)} or Hes5 promoter^{37 (link)} was subcloned into vector CSII^{51 (link)}. Lentiviral particles were produced in HEK 293T cells cultured in OptiPRO serum-free medium (Gibco) and concentrated by centrifugation after washing with PBS. Viruses were delivered stereotactically into the DG of 6-week-old C57BL/6 male mice using the following coordinates: 1.9 mm posterior to bregma, 1.4 mm lateral to the midline, and 1.75 mm below the dura.

Kobayashi T., Piao W., Takamura T., Kori H., Miyachi H., Kitano S., Iwamoto Y., Yamada M., Imayoshi I., Shioda S., Ballabio A, & Kageyama R. (2019). Enhanced lysosomal degradation maintains the quiescent state of neural stem cells. Nature Communications, 10, 5446.

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Characterization of Murine 4-1BB Binding

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The gene for murine 4-1BB (OriGene) was cloned into the pIRES2 expression vector, which encodes for GFP downstream of the inserted 4-1BB gene using an IRES site, using In-Fusion cloning (Takara Bio). Freestyle 293-F cells were transiently transfected by mixing 1 mg/mL of plasmid DNA and 2 mg/mL of polyethylenimine (Polysciences) in OptiPRO Serum Free Medium (Gibco) and, after incubating, adding dropwise to the cells. 3-5 days after transfection, cells were harvested and pelleted in V-bottom 96 well plates. Cells were titrated with ɑ4-1BB or ɑ4-1BB-LAIR and incubated for 3 h shaking at 4 °C. Cells were washed with PBSA (PBS (Corning) + 0.1% BSA (Sigma Aldrich)) and incubated with ɑmIgG1-APC (diluted 1:250, clone M1-14D12, Invitrogen) for 30 min shaking at 4 °C. Data were collected on a BD LSR II cytometer (BD Biosciences). Binding curves were generated with GraphPad Prism software V9. K_D values were calculated using a nonlinear regression fit for one site total binding with no non-specificity and curves were normalized to the B_max values.

Palmeri J.R., Lax B.M., Peters J.M., Duhamel L., Stinson J.A., Santollani L., Lutz E.A., Pinney W I.I.I., Bryson B.D, & Dane Wittrup K. (2024). CD8+ T cell priming that is required for curative intratumorally anchored anti-4-1BB immunotherapy is constrained by Tregs. Nature Communications, 15, 1900.

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Propagation of E1-Transformed MDCK Cells

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The E1-transformed MDCK cell line, previously established at our lab12 (link) by transforming MDCK cells (ECCAC Ref. 84121903) with the E1 region from Canine Adenovirus type 2, was cultured in Optipro serum-free medium (Gibco) containing 4 mM glutamine. Dog Kidney cells expressing the same E1 protein (DKZeo8 (link)) were used for viral stock preparation and titration, and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Gibco). Both cell lines were routinely maintained in T-flasks, in a humidified incubator at 5% CO₂ and 37 °C, and sub-cultured twice a week.

Carinhas N., Pais D.A., Koshkin A., Fernandes P., Coroadinha A.S., Carrondo M.J., Alves P.M, & Teixeira A.P. (2016). Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production. Scientific Reports, 6, 23529.

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