Freestyle max transfection reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States

The FreeStyle Max transfection reagent is a laboratory product designed for the efficient delivery of nucleic acids into mammalian cells. It facilitates the transfection process, enabling the introduction of genetic material into cells.

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Transfection reagent (92 citations) FreeStyle MAX Reagent (53 citations) Freestyle MAX (11 citations)

15 protocols using freestyle max transfection reagent

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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Optimized Expression and Purification of SARS-CoV-2 Proteins

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The expression vector of the S trimer was transfected into the CHO-S cell line according to the manufacturer’s instructions. Briefly, CHO-S cells were seeded at 6 × 10⁵ cells/mL in Forti-CD CHO medium (Gibco, Shanghai, China, A11483) and incubated in an orbital shaking incubator at 37 °C and 120 rpm with 8% CO₂ overnight. The next day, 1.0 × 10⁶ cells/mL were transfected using FreeStyleMax transfection reagent (Thermo Fisher, Waltham, MA, USA, 16447100) and grown for five days at 35 °C. The cell culture medium was harvested by centrifuge at 4800 rpm to remove cells. The supernatant was collected and passed through a 0.45 μm filter and then loaded onto the pre-equilibrated Ni Smart Beads 6FF (Changzhou Smart-Lifesciences Biotechnology Co., Ltd., Changzhou, China, SA036100) affinity column for protein purification. The eluted proteins were then buffer exchanged to PBS by ultracentrifugation using 50 kDa ultrafiltration cubes (Cytiva, Shanghai, China, 28932362).
The S1-D614G, Omicron S trimer, E484K RBD, N501Y RBD, Beta RBD, Delta RBD, and Gamma RBD proteins used in this study were purchased from Sino Biological (Beijing, China) or Novoprotein (Shanghai, China), and detailed information is listed in Supplementary Table S1.

Wang H., Wang Z., Ma L., Zhu X., Li B., Huang Y., Li J., Sun M., Shi L, & Yao Y. (2023). S Trimer Derived from SARS-CoV-2 B.1.351 and B.1.618 Induced Effective Immune Response against Multiple SARS-CoV-2 Variants. Vaccines, 11(1), 193.

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Efficient Induction of Apoptosis in CHO Cells

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CHO-S cells (Thermo Fisher Scientific) were maintained in CD CHO medium supplemented with 8 mM L-Glutamine (Thermo Fisher Scientific) and 2 μL/mL Anti-Clumping reagent (AC, Life Technologies). Cells were cultivated in 125 mL Erlenmeyer shake flasks (Corning Inc., Acton, MA), incubated in a humidified incubator at 37˚C, 5% CO₂ at 120 rpm and passaged every 2-3 days. One day before transfection, cells were seeded into the culture medium without AC. On the day of transfection, cells were transfected using a total amount of 3.75 μg of DNA packaged with 3.75 μL of FreeStyle Max transfection reagent (Thermo Fisher Scientific) per well of a 6-well plate (BD Biosciences) in 3 mL culture medium without AC at the adjusted density of 1.0 × 10⁶ viable cells/mL (details about ratio of gRNA and dCas9 repressor for transfection in Supplementary table S3). All transfections were performed in biological duplicates or triplicates. To induce apoptosis, 10 μg/mL of ActD (Sigma Aldrich) was added into the culture of KRAB-dCas9-CHO cells at 24 hours after transfection with or without gRNA. 24 hours post ActD induction, cells were harvested for detection of mitochondrial membrane potential and caspase activity as described below.

Xiong K., Marquart K.F., la Cour Karottki K.J., Li S., Shamie I., Lee J.S., Gerling S., Yeo N.C., Chavez A., Lee G.M., Lewis N.E, & Kildegaard H.F. (2019). Reduced Apoptosis in Chinese Hamster Ovary Cells via Optimized CRISPR Interference. Biotechnology and bioengineering, 116(7), 1813-1819.

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Transfection of CHO-S Cells for CRISPR Screening

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CHO-S cells (Thermo Fisher Scientific) were cultured in CD CHO medium supplemented with 8 mM L-Glutamine (Thermo Fisher Scientific) and 2 μL/mL Anti-Clumping reagent (AC, Life Technologies). Cells were maintained in Erlenmeyer shake flasks (Corning Inc., Acton, MA), incubated in a humidified incubator at 37°C, 5% CO2 at 120 rpm and passaged every 2-3 days. Viable cell density (VCD) and viability were monitored using the NucleoCounter NC-200 Cell Counter (ChemoMetec, Denmark). One day before transfection, cells were seeded into the culture medium without AC. On the day of transfection, cells were transfected using 3.75 μg of DNA packaged with 3.75 μL of FreeStyle Max transfection reagent (Thermo Fisher Scientific) per well of a 6-well plate (BD Biosciences) in 3 mL culture medium without AC and with 1E6 cells/mL. Specifically, for VF gRNA library transfection, 150 μg Bxb1 recombinase plasmid and 450 μg gRNA library donor were transfected into 480 mL CHO-attp-mCherry cells at the density of 1E6 cells/mL. The cells were divided into several 6-well plates and one day after transfection, the cells were combined into two 500 mL flasks. Transfection efficiencies were monitored using the transfection reagent included pmax-GFP plasmid and was generally observed to be between 60-90%.

Xiong K., la Cour Karottki K.J., Hefzi H., Li S., Grav L.M., Li S., Spahn P., Lee J.S., Ventina I., Lee G.M., Lewis N.E., Kildegaard H.F, & Pedersen L.E. (2021). An optimized genome-wide, virus-free CRISPR screen for mammalian cells. Cell Reports Methods, 1(4), 100062.

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Transient Antibody Expression in FreeStyle 293-F Cells

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FreeStyle™ 293-F cells (Catalog number R79007, Thermo-Fisher Scientific, Waltham, MA) in suspension were maintained in FreeStyle™ 293 expression media in 125 ml Erlenmeyer flasks on an orbital shaker (135 rpm) in 8% CO₂ at 37 °C according to the manufacturer’s instructions (Thermo-Fisher Scientific, Waltham, MA). Cells (30 ml at 1 × 10⁶ cells ml⁻¹) in flasks were transiently transfected with antibody expression vectors (pVITRO-Cat IgG-IgL; pVITRO-Cat IgG-IgK; or pVITRO-felinized mAb E) using FreeStyle™ MAX transfection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Transfected cells were collected on day 7, centrifuged at 1,000 × g for 15 min, then the supernatants were filtered through 0.2 μm filters (Whatman, Shrewsbury, MA).
Recombinant antibodies were purified by loading Ig-containing supernatants onto a 1 ml HiTrap Protein A HP column. After washing in phosphate buffered saline (PBS), purified antibodies were eluted with 0.1 M glycine-HCl buffer (pH 2.7), and fractions were collected into tubes containing 1 M Tris-HCl (pH 9).

Lu Z., Tallmadge R.L., Callaway H.M., Felippe M.J, & Parker J.S. (2017). Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus. Scientific Reports, 7, 12713.

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Engineered CHO-C1 Cell Line for Glycosylation Studies

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CHO-S cells (Thermo Fisher Scientific) and a CHO-S mutant with knockouts of Mgat4a,4b and 5, St3gal3,4 and 6, B3gnt2, Sppl3, and Fut8 (referred to here as CHO-C1) were maintained in CD-CHO medium supplemented with 8mM L-glutamine (Thermo Fisher Scientific) and 1 μL/mL Anti-Clumping (AC) reagent (Life Technologies), unless otherwise specified. The cells were cultivated in 125mL Erlenmeyer shake flasks (Corning) in a humidified incubator at 37˚C, 5% CO₂ at 120 RPM and passaged every 2–3 days. Viable cell density (VCD) and viability were monitored using the Nucleocounter NC-200 Cell Counter (ChemoMetec). One day prior to transfection, 0.6 × 10⁶ cells/mL cells were spun down at 200x g and resuspended in 6-well plates (BD Biosciences) in 3 mL culture medium without AC per well. Cells were transfected using a total of 3.75 μg DNA and 3.75 μL FreeStyle™ Max transfection reagent (Thermo Fisher Scientific) together with OptiPRO SFM (Life Technologies) according to manufacturer’s protocol. The ratio of DNA used from respective plasmids is listed in Supplementary Table S4. Plasmids for gRNAs targeting Aldh18a1 were transfected into CHO-S cells; those targeting Mgat3 and St6gal1 were transfected into CHO-C1 cells. In addition, non-targeting gRNA with and without dCas9 were transfected for each cell line as controls. All transfections were done in three biological replicates.

Karottki K.J., Hefzi H., Xiong K., Shamie I., Hansen A.H., Li S., Pedersen L.E., Li S., Lee J.S., Lee G.M., Kildegaard H.F, & Lewis N.E. (2019). Awakening dormant glycosyltransferases in CHO cells with CRISPRa. Biotechnology and bioengineering, 117(2), 593-598.

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Efficient Transfection of CHO-S Cells

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CHO-S cells (Thermo Fisher Scientific) were cultured in CD CHO medium supplemented with 8 mM L-Glutamine (Thermo Fisher Scientific) and 2 μL/mL Anti-Clumping reagent (AC, Life Technologies). Cells were maintained in Erlenmeyer shake flasks (Corning Inc., Acton, MA), incubated in a humidified incubator at 37°C, 5% CO2 at 120 rpm and passaged every 2–3 days. Viable cell density (VCD) and viability were monitored using the NucleoCounter NC-200 Cell Counter (ChemoMetec, Denmark). One day before transfection, cells were seeded into the culture medium without AC. On the day of transfection, cells were transfected using 3.75 μg of DNA packaged with 3.75 μL of FreeStyle Max transfection reagent (Thermo Fisher Scientific) per well of a 6-well plate (BD Biosciences) in 3 mL culture medium without AC and with 1E6 cells/mL. Specifically, for VF gRNA library transfection, 150 μg Bxb1 recombinase plasmid and 450 μg gRNA library donor were transfected into 480 mL CHO-attp-mCherry cells at the density of 1E6 cells/mL. The cells were divided into several 6-well plates and one day after transfection, the cells were combined into two 500 mL flasks. Transfection efficiencies were monitored using the transfection reagent included pmax-GFP plasmid and was generally observed to be between 60–90%.

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Preparation and Characterization of eeIFSH

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The pGEM-T easy cloning vector was purchased from Promega (Madison, WI, USA). The pCORON1000 SP VSV-G tag expression vector was purchased from Amersham Biosciences (Piscataway, NU, USA). The pcDNA3 expression vector, FreeStyle™ MAX transfection reagent, Lipofectamine-3000, and FreeStyle CHO-suspension (CHO-S) cells were provided by Invitrogen (Carlsbad, CA, USA). Ham’s F-12 medium, and OptiMEM medium were purchased from Gibco BRL (Grand Island, NY, USA). CHO-K1 and HEK 293 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). A homogeneous time-resolved fluorescence (HTRF) cAMP assay kit was purchased from Cisbio (Codolet, France). Monoclonal antibodies (5A11, 11A8, and 14F5) used in the ELISA analysis and eelFSH from CHO-S cells were produced in our lab as previously reported [41 (link)]. The horseradish peroxidase (HRP) labeling of 11A8 and 14F5 monoclonal antibodies was generously performed by Medexx Inc. (Seongnam, Korea). QIAGEN Maxi plasmid kits were purchased from Qiagen Inc. (Hilden, Germany). The glass spinner flasks and disposable flasks were provided by Corning Inc. (Corning, NY, USA). All other reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Chemicals (Osaka, Japan).

Byambaragchaa M., Kim J.S., Park H.K., Kim D.J., Hong S.M., Kang M.H, & Min K.S. (2020). Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor. International Journal of Molecular Sciences, 21(19), 7075.

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