Gibco opti mem

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

Gibco Opti-MEM is a cell culture medium designed for the maintenance and growth of a variety of cell lines. It is a serum-free and protein-free formulation that provides essential nutrients and growth factors to support cell proliferation and viability in cell culture applications.

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GibcoTM Opti-MEMTM Gibco® Opti-MEM™ medium Gibco™ Opti-MEM™ GlutaMAX Gibco® Opti-MEM™ Media Gibco Opti-MEM reduced serum media GIBCO™ Opti-MEM I (GlutaMAX™-I) Reduced-Serum Medium liquid Gibco Opti-MEM I Reduced Serum Media Gibco Opti-MEM I Reduced Serum Medium Gibco's Opti-MEM Reduced Serum Medium Gibco Opti-MEM reduced serum medium

22 protocols using gibco opti mem

Flavivirus Neutralization Titer Assay

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We determined neutralization titers against DENV and ZIKV by focus-reduction neutralization test (FRNT) in a 96-well format described elsewhere (24 (link)). Serially diluted serum was mixed with 50–100 focus-forming units of the virus in Dulbecco modified Eagle medium with 2% fetal bovine serum. We incubated the antibody and virus complexes (1 h, 37°C), then transferred them to a monolayer of Vero-81 cells for infection. After 1 additional hour of incubating antibody and virus complex on Vero-81 monolayer, we overlaid cells with GIBCO Opti-MEM (https://www.thermofisher.com), a modified Eagle medium containing 2% fetal bovine serum and 1% carboxymethylcellulose. After allowing the predetermined time required to form viral foci, we fixed Vero-81 cells and immunostained them with Flavivirus-specific monoclonal antibodies. For neutralization assays, we calculated 50% inhibitory concentration by using the sigmoidal dose-response (variable slope) equation in Prism 6 (GraphPad Software, https://www.graphpad.com). For the study, we included only reported values with an R² (coefficient of determination) >0.75, hill slope >0.5, and 50% inhibitory concentration within the assay range. We performed FRNT for Flavivirus strains WP74 (DENV-1), S16803 (DENV-2), CH53489 (DENV-3), TVP-376 (DENV-4), and H/PF/2013 (ZIKV).

Adams C., Jadi R., Segovia-Chumbez B., Daag J., Ylade M., Medina F.A., Sharp T.M., Munoz-Jordan J.L., Yoon I.K., Deen J., Lopez A.L., de Silva A.M, & Premkumar L. (2021). Novel Assay to Measure Seroprevalence of Zika Virus in the Philippines. Emerging Infectious Diseases, 27(12), 3073-3081.

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Plasmid DNA Transfection in Cells

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Cells were twice washed in Dulbecco’s phosphate-buffered saline (DPBS, Gibco Thermo Fisher Scientific) and then collected in R buffer with a density of 2 × 10⁷ cells/ml. For electroporation, we used 5 µg plasmid DNA (with a concentration of at least 1 µg/µl) to 2 × 10⁶ cells in R buffer for a 100 µl NEON electroporation pipette tip (Thermo Fisher Scientific) at 1600 V for 30 ms and 1 pulse. Cells were harvested 24 h later. For validation experiments (Fig. 3e), 7 × 10⁵ cells per well were seeded in six-well TC plates one day prior to transfection. 24 h later, cells were transfected with a mixture of 2 µg plasmid DNA and 20 µg linear Polyethylenimine MW 2500 (PEI 2500, Polysciences), and Gibco Opti-MEM (Thermo Fisher Scientific) was added to 100 µl total volume. This mixture was added dropwise to 1,9 ml fresh DMEM covering the cells, followed by incubation for 24 h.

Cortés-López M., Schulz L., Enculescu M., Paret C., Spiekermann B., Quesnel-Vallières M., Torres-Diz M., Unic S., Busch A., Orekhova A., Kuban M., Mesitov M., Mulorz M.M., Shraim R., Kielisch F., Faber J., Barash Y., Thomas-Tikhonenko A., Zarnack K., Legewie S, & König J. (2022). High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance. Nature Communications, 13, 5570.

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Transfection of EGFP-ApAFP752 in HEK 293T Cells

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Plasmids for EGFP and EGFP–ApAFP752 were designed and purchased from GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). Transfection of EGFP–ApAFP752 plasmid (Figure S1) into HEK 293T cells was optimized according to manufacturer protocols. After optimization, 1 × 10⁷ cells were seeded into T75 flasks with a final volume of 19.7 mL DMEM supplemented with 10% FBS and incubated overnight. The following day, cells were ~80% confluent. A volume of 20 µL of plasmid DNA (1 µg/µL) was combined with 2 mL of Gibco™ OptiMEM (Thermo Fisher Scientific, Waltham, MA, USA), then 60 µL of TransIT^®-293 (Mirus Bio, Madison, WI, USA) was mixed in and left for 30 min at room temperature to complex. The solution was then mixed, added to the ~80% confluent T75 flask of HEK 293T cells, and cells were incubated for 48 h. Following incubation, cells were observed, and digital images were taken with epifluorescence microscopy to visualize EGFP and EGFP–ApAFP752 transfection.

Sreter J.A., Foxall T.L, & Varga K. (2022). Intracellular and Extracellular Antifreeze Protein Significantly Improves Mammalian Cell Cryopreservation. Biomolecules, 12(5), 669.

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Knockdown of HOX13 Genes in Acral Melanoma Cell Lines

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Three human acral melanoma cell lines including SKMEL-1088, SKMEL-1176 and SKMEL-1206 were used for knockdown of four HOX13 genes. Non-targeting Control siRNA (D-001810–01-05), HOXA13 siRNA SMARTPool (L-011052–00-0005), HOXB13 siRNA SMARTPool (L-012226–00-0005), HOXC13 siRNA SMARTPool (L-017600–00-0005) and HOXD13 siRNA SMARTPool (L-011053–00-0005) were purchased from Horizon Discovery. In brief, 4 × 10⁵ cells were seeded in 6-well plates and cultured with RPMI 1640 medium (ThermoFisher 11875093) containing 10% FBS (Seradigm), 1× PSG (Gibco 10378016) for 24 h. Medium was then replaced with 1 ml of fresh medium before transfection. One hundred nanomolar total siRNA (25 nM of each hox siRNA) was added to 200 μl Gibco Opti-MEM (ThermoFisher 31985070) and mixed with 12 μl DharmaFECT Duo Transfection Reagent (Horizon Discovery #T-2010–03) followed by a 10 s of agitation. After 10 min of incubation at room temperature, transfection mixtures were evenly dropped into each well. Media was aspirated 6 h later and replaced with 2 ml fresh RPMI medium containing 10% FBS, 1× PSG. Forty-eight hours after transfection, cells were changed to 3 ml of RPMI, 20% FBS, 1× PSG for western blot or 1.5 ml of serum-free RPMI, 1× PSG for ELISA. Samples were collected 96 h after transfection for both western blot and ELISA.

Weiss J.M., Hunter M.V., Cruz N.M., Baggiolini A., Tagore M., Ma Y., Misale S., Marasco M., Simon-Vermot T., Campbell N.R., Newell F., Wilmott J.S., Johansson P.A., Thompson J.F., Long G.V., Pearson J.V., Mann G.J., Scolyer R.A., Waddell N., Montal E.D., Huang T.H., Jonsson P., Donoghue M.T., Harris C.C., Taylor B.S., Xu T., Chaligné R., Shliaha P.V., Hendrickson R., Jungbluth A.A., Lezcano C., Koche R., Studer L., Ariyan C.E., Solit D.B., Wolchok J.D., Merghoub T., Rosen N., Hayward N.K, & White R.M. (2022). Anatomic position determines oncogenic specificity in melanoma. Nature, 604(7905), 354-361.

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Transient Plasmid Transfection in 24-well Format

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Cells were transfected at early post-defrosting passages (P5-P15) when reaching 30–50% confluency. Assays were performed in a 24-well plate system (Corning). A total of 0.5 μg of plasmids and 2 μl of Lipofectamine 2000 were used at a final transfection volume of 100 μl. Gibco™ Opti-MEM™ (Thermo Fisher Scientific) was used as transfection medium. RNA or protein were isolated at 48 h following transfection.

Gomes-Duarte A., Venø M.T., de Wit M., Senthilkumar K., Broekhoven M.H., van den Herik J., Heeres F.R., van Rossum D., Rybiczka-Tesulov M., Legnini I., van Rijen P.C., van Eijsden P., Gosselaar P.H., Rajewsky N., Kjems J., Vangoor V.R, & Pasterkamp R.J. (2022). Expression of Circ_Satb1 Is Decreased in Mesial Temporal Lobe Epilepsy and Regulates Dendritic Spine Morphology. Frontiers in Molecular Neuroscience, 15, 832133.

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FADD Knockdown Effects on H1299 Proliferation

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GenePharma Co., Ltd. generated both FADD knockdown plasmids and control plasmids. H1299 (5 × 10⁵) cells on a 6-well plate were plated and cultured at 37°C overnight using RPMI-1640. These cells were incubated in serum-free Gibco™-OptiMEM (from Thermo Fisher Scientific, Inc.) for 6 h at 37°C after being treated with shRNA and Lipofectamine^®2000 Reagent (from Invitrogen, Thermo Fisher Scientific, Inc.). The cells were then added with Fresh RPMI-1640 containing 10% FBS. For the effect of FADD on the proliferation of H1299, the concentrations of FADD siRNA were 0, 10, 25, 50, 100, and 200 nM, and the concentrations of the total shRNA were made up to 200 nM with NC shRNA for those transfections with a FADD shRNA concentration <200 nM. After 48 h of shRNA transfection, H1299 cells were treated with Adriamycin to observe the effect of FADD on the cells’ drug resistance, where the concentration of FADD shRNA used was 100 nM.

Wei S., Chen Z., Ling X., Zhang W, & Jiang L. (2023). Comprehensive analysis illustrating the role of PANoptosis-related genes in lung cancer based on bioinformatic algorithms and experiments. Frontiers in Pharmacology, 14, 1115221.

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Transfection of Cardiomyocytes Using siRNA

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Cardiomyocytes were seeded at equal number of cells (2.0 × 10⁵ per dish) in 35 mm petri dish and maintained in the absence of antibiotic culture medium for 24 h before transfection, then washed three times with PBS. Cardiomyocytes were transfected with Control siRNA (Con-siRNA) and gp78-siRNA (Santa, Dallas, TX, USA) using Lipofectamine™ 2000 transfection reagent from Invitrogen™ (Thermo Fisher Scientific, Scotland, UK). siRNA and the transfection reagent complex were added to the reduced serum media (Gibco™ Opti-MEM™, Thermo Fisher Scientific, UK) for 8 h, the transfection continued for another 24 h in serum-containing regular medium. After that, the cells were subjected to research.

Wang Y., Gao P., Wei C., Li H., Zhang L., Zhao Y., Wu B., Tian Y., Zhang W., Wu L., Wang R, & Xu C. (2017). Calcium sensing receptor protects high glucose-induced energy metabolism disorder via blocking gp78-ubiquitin proteasome pathway. Cell Death & Disease, 8(5), e2799-.

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Transfecting Breast Cancer Cells with DSCAM-AS1 siRNA

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Chemically synthesized siRNAs were purchased from RiboBio (Guangzhou, China). siRNA target sequences for DSCAM‐AS1 were as follows: si‐DSCAM‐AS1‐1, 5′‐GGAGATCACAGCCAAGGAA‐3′ and si‐DSCAM‐AS1‐2, 5′‐GCATGACATACTCATCCAT‐3′. For RNA transfection, 3 × 10⁵ (MCF‐7) or 5 × 10⁵ (T‐47D) cells were seeded onto 6‐well plates. Twenty‐four hours after seeding, 60 nmol/L siRNA was transfected using Lipofectamine^® RNAiMAX (ThermoFisher Scientific, 13778150) and Gibco^™ Opti‐MEM (ThermoFisher Scientific, 51985042) according to the manufacturer's recommendations.

Sun W., Li A., Zhou P., Jiang Y., Jin X., Liu Y., Guo Y., Yang W., Shao Z, & Xu X. (2018). DSCAM‐AS1 regulates the G1/S cell cycle transition and is an independent prognostic factor of poor survival in luminal breast cancer patients treated with endocrine therapy. Cancer Medicine, 7(12), 6137-6146.

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Silencing PNPLA2 Gene in ARPE-19 Cells

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Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). Their sequences and that of a scramble siRNA (Scr) (SR324651 and SR311349) are given in Table 3. From the six duplexes, siRNAs C, D, and E consistently provided the highest silencing efficiency; therefore, these three duplexes were used individually for silencing experiments and are referred to as siPNPLA2. ARPE-19 cells were transfected by reverse transfection in 24-well tissue culture plates as follows: A total of 6 pmol of siRNA was diluted in 100 µL of Gibco OptiMem (Thermo Fisher Scientific) per well and mixed with 1 µL of Invitrogen Lipofectamine RNAiMAX; mock transfected cells received only 1 µl of Lipofectamine. The mixture was then added to each well. After incubation at room temperature for 10 minutes, a total of 1 × 10⁵ cells in 500-µL antibiotic-free DMEM/F-12 containing 10% FBS was added to each well, and the plate was swirled gently to mix. Assays were performed 72 hours after transfection.

Bullock J., Polato F., Abu-Asab M., Bernardo-Colón A., Aflaki E., Agbaga M.P, & Becerra S.P. (2021). Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R). Investigative Ophthalmology & Visual Science, 62(2), 30.

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Colon Cancer Cell Transfection Protocol

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Glutamine, DMEM, trypsin-EDTA, and penicillin/streptomycin were obtained from GE Healthcare HyClone (Logan, UT, United States). Fetal bovine serum, enzymes, 3-aminopropyltriethoxy silane (TESPA), L-15 and chemicals were from Sigma (St. Louis, MO, United States). DiI-C₁₂, DiD-C₁₈, Lipofectamine 2000 and Gibco Opti-MEM were from Thermo Fisher Scientific (Waltham, MA, United States). High performance, 1.5H, coverslips were from Marienfeld (Lauda-Königshofen, Germany). The colon cancer cell line HT29 was a kind gift of Dr. A. Blokzijl, Uppsala University, Sweden and SW480 cells were from ATCC (Manassas, VA, United States). CD3ζ-EYFP, mouse CD3ζ fused to EYFP via a six aa linker expressed in the pBJ1-Neo plasmid under the CMV-promotor, was from Mark Davis, Stanford University School of Medicine, United States (Krummel et al., 2000 (link)). Lck-EGFP, a fusion protein of mouse Lck and EGFP via a six aa linker expressed in the pcDNA3 under the CMV-promotor, was from Tony Magee, Imperial College London, United Kingdom (Janes et al., 1999 (link)). Borosilicate glass (1B100F-4) was from World Precision instruments (Sarasota, FL, United States).

Gesper A., Wennmalm S., Hagemann P., Eriksson S.G., Happel P, & Parmryd I. (2020). Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy. Frontiers in Cell and Developmental Biology, 8, 767.

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