Opti memtm

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Opti-MEMTM is a cell culture medium designed to support the growth and maintenance of a variety of cell lines. It is a serum-free, low-protein formulation that provides essential nutrients and growth factors to help maintain cell viability and proliferation.

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Opti-MEM (8992 citations) Opti-MEMTM medium (13 citations) Gibco Opti-MEM reduced serum medium (10 citations) Opti-MEMTM reduced serum medium (9 citations) Opti-MEMTM I Reduced Serum Medium (7 citations) Opti-MEMTM media (4 citations) GibcoTM Opti-MEMTM (3 citations) Opti-MEM I solution (3 citations) Opti-MEMTM Reduced Serum Media (2 citations) Opti-MEMTM I reduced serum media (2 citations)

50 protocols using opti memtm

Transfection and Silencing of TIMAP and MYPT1

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For transfection with His₆- or GFP-TIMAP cDNA, COS7 cells in the logarithmic phase of replication were transfected with Lipofectamine 2000® (Life Technologies) and 4 μg of plasmid cDNA per 35-mm plate. Cells were used 48 h after transfection. His₆- and GFP-TIMAP protein expression was verified by WB analysis.
To silence TIMAP or MYPT1 expression, ECs were plated in 35-mm culture dishes at ∼60% confluence. 24 h later, the medium was replaced with 1 ml of EBM-2 lacking antibiotics. 60 pmol of control, 60 pmol of hTIMAP-specific siRNA, or 30 and 60 pmol of control or hMYPT1-specific siRNA were added to 100 μl of Opti-MEM^TM (Gibco) combined with 6 μl of Lipofectamine 3000® in 100 μl of Opti-MEM^TM, incubated for 5 min at room temperature, and then added to 1.0 ml of EBM-2 medium in each 35-mm dish. 6 h later, 1.0 ml of EBM-2 medium containing 10% FBS (no antibiotics) was added, and the cells were harvested 48 h after addition of siRNA.

Wang X., Obeidat M., Li L., Pasarj P., Aburahess S., Holmes C.F, & Ballermann B.J. (2019). TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ. The Journal of Biological Chemistry, 294(36), 13280-13291.

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Lipoplex Formulation for GFP Transfection

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All lipoplex formulations were prepared following the manufacturer’s recommended protocol. Briefly, we first prepared the two pre-solutions: 3.6 μl of Lipofectamine 3000 reagent mixed into 60 μl of Opti-MEM^TM by pipetting (pre-solution 1), and 4.8 μl of P3000 reagent along with 2,400 ng of pGFP mixed into 60 μl of Opti-MEM^TM (Fisher Scientific) by pipetting (pre-solution 2). Lipoplexes were formed by mixing pre-solution 1 and 2, followed by incubation for 15 min at room temperature before use.

Ayyadevara V.S, & Roh K.H. (2020). Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells. Drug Delivery, 27(1), 805-815.

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FUS and TDP-43 aggregation dynamics

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Cells were seeded on poly-L-lysine−coated coverslips (2.5 × 10⁵ cells) one day prior to the experiment. When ∼ 50% confluency was reached, cells were transiently transfected with 1 µg FUS-GA, 1 µg FUS-B, and 1 µg TDP-43-RA via 6 µl lipofection (X-TremeGene 9, Roche) in 300 µl Opti-MEM^TM (Life Technologies), or 1 µg tandem plasmid CMV-FUS-GA-IRES-FUS-B-IRES-TDP-43-RA via 2 µl lipofection in 100 µl Opti-MEM^TM. Proteins were transiently expressed for 24 h at 37 °C under 5% CO₂ in a HERAcell Vios 160i LK CO₂ incubator (Thermo Fisher Scientific). Cells were treated with 25 µM AdOx (Selleck Chem) for 24 h and stained with 1 µM Hoechst 33342 (Enzo) for 10 min before confocal imaging. Cells transfected with 1 µg TDP-43-GA and 1 µg TDP-43-B or 1 µg TDP-43-eGFP only were treated with 5 µM CuET (TCI) for 3 h before imaging. All confocal images of cells were got with Leica Stellaris 5 High-resolution Laser Confocal Fluorescence Microscope.

Huang Y., Chen J., Hsiung C.H., Bai Y., Tan Z., Ye S, & Zhang X. (2024). Detecting protein−protein interaction during liquid−liquid phase separation using fluorogenic protein sensors. Molecular Biology of the Cell, 35(3), ar41.

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In vitro Transwell Migration Assay

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In vitro migration assays were carried out using 6.5 mm Transwell plates with 5.0 μm pore polycarbonate membrane inserts (Corning, Torino, Italy). Uninfected and infected THP-1 cells and primary human monocytes (in both cases 10⁵ cells) were suspended in 100 µL serum-free medium (OptiMEM^TM, Gibco) and seeded in the inserts, while 600 µL of serum-free medium alone or serum-free medium in which cancer cells had been cultured were placed in the well. Transwell plates were incubated for 3 h at 37 °C in a 5% CO₂ atmosphere with 98% humidity. Inserts were removed and migrated cells were stained with CellTracker™ Red CMTPX Dye (Invitrogen) following the manufacturer’s instructions and were counted over at least three different 10× microscopy fields.

Reale A., Krutzke L., Cadamuro M., Vitiello A., von Einem J., Kochanek S., Palù G., Parolin C, & Calistri A. (2023). Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer. International Journal of Molecular Sciences, 24(11), 9255.

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Overexpression and Knockdown of Circular RNAs

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To construct the overexpression plasmids of circPTPRA, LMNB1, EIF4A3, and FUS, the synthetic human circPTPRA cDNA was cloned into the pLC5‐ciR vector by Geneseed Biotech Co., Ltd., the synthetic human LMNB1, EIF4A3, and FUS cDNAs were cloned into the pEX‐3 vector by GenePharma, and the empty plasmid was used as a control. The siRNA sequences of circPTPRA, LMNB1, EIF4A3, and FUS, as well as the mimics and inhibitor of miR‐140‐5p were designed and synthesized by RiboBio Co., Ltd. Lipofectamine^TM 3000 (Invitrogen) and Opti‐MEM^TM (Gibco) were used as transient transfection reagents according to the manufacturer's instruction. Total RNA was extracted 48 h after transfection. The sequences of siRNAs, sh‐RNAs, plasmids, mimics and inhibitors are shown in Additional file 1 Table S1.
The overexpression and knockdown lentiviruses of circPTPRA and LMNB1 were constructed by Genechem Co., Ltd. Lentiviral infection was performed according to the manufacturer's instruction. Stable transfected cells were selected by culturing in medium containing 5 μg/mL puromycin (Beyotime), and the expression of circPTPRA and LMNB1 was confirmed by quantitative real‐time PCR (qRT–PCR).

Fu W., Wang X., Xiang J., Chen S., Xia R., Xie F., Chi B., Qin F., Li Z., Mou L., Xie C, & Wang H. (2023). CircPTPRA promotes the progression of pancreatic ductal adenocarcinoma via the miR‐140‐5p/LMNB1 axis. Cancer Medicine, 12(10), 11651-11671.

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FASN Knockdown in Cell Cultures

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Cells were seeded in 60 mm dishes at a density of 5 × 10⁴ cells/ml. 24h post-seeding, cells were transfected with siRNA against fatty acid synthase (FASN, Santa Cruz, SC-41516, 17 pmol), using lipofectamine (RNAiMAX, ThermoFisher) as the transfection reagent and Opti-MEM^TM (Gibco^TM) as the transfection medium. Control siRNA (Qiagen, 1027280) was used as a negative control. Cells were harvested 24h post-transfection and seeded into appropriate dishes for further study. Transfection efficiency was determined via immunoblotting against FASN (1/1000, Cell Signaling Technology, #3189).

Ates G., Goldberg J., Currais A, & Maher P. (2020). CMS121, a fatty acid synthase inhibitor, protects against excess lipid peroxidation and inflammation and alleviates cognitive loss in a transgenic mouse model of Alzheimer's disease. Redox Biology, 36, 101648.

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Transfection and RNA/IF Imaging

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Patient-derived clones were transfected in HEK 293T cells in a 1:3 dilution of DNA: Fugene (Promega, United States) using 1 μg DNA for 24 h. All transfection reagents were mixed in Opti-MEM^TM (Gibco, Life Technologies, Carlsbad, United States) to a proportion of 1:10 of total media volume of RPMI. After 24 h post-transfection, media was removed and cells washed twice in PBS prior fixation using 10% NBF for 20 min at RT. Subsequently, RNAscope was performed and IF using primary and secondary antibody staining. Images were acquired using single point scanning confocal microscopy.

Zhang W., Svensson Akusjärvi S., Sönnerborg A, & Neogi U. (2018). Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution. Frontiers in Microbiology, 9, 2358.

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Extracellular Vesicle Purification and Characterization

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EVs were purified according to differential centrifugation protocols, with slight modification [71 ]. WT BMDM and gorasp2^−/- BMDM from C57BL/6 were seeded in culture dishes in complete DMEM containing 8% L929 culture supernatant, at approximately 70% confluence. After washing with DPBS (Gibco, A1285801), the cells were incubated in OPTI-MEM^TM (Gibco, 31,985,088) with 250 ng/mL LPS for 24 h. In WT MEF and atg5^−/- MEF cells were washed with DPBS and incubated in EBSS medium for 14 h with 20 nM Baf and 20 μM CQ. The supernatants were harvested and centrifuged at 300 × g for 10 min to remove the cell debris. The supernatants were centrifuged at 2,000 × g for 20 min to remove apoptotic bodies and then at 10,000 × g for 30 min to remove large particles. Finally, the supernatants were ultracentrifuged at 100,000 × g for 2 h, and the pellets (EVs) were resuspended in DPBS. Protein concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific, 23,225). The same amount of EVs from each sample was incubated with 20 μg/mL proteinase K (New England BioLabs, P8107S), with or without 1% Triton X-100, for 1 h at 37°C. The reaction was stopped by adding 2× protein sample buffer, and the lysates were immunoblotted.

Kim Y.H., Kwak M.S., Lee B., Shin J.M., Aum S., Park I.H., Lee M.G, & Shin J.S. (2020). Secretory autophagy machinery and vesicular trafficking are involved in HMGB1 secretion. Autophagy, 17(9), 2345-2362.

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Packaging and Transducing Retro- and Lentiviruses

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Retrovirus and lentivirus were packaged as follows. 293FT cells were plated on 6-well plates at roughly 50% confluence in full-growth medium and 16–24 h later were transfected with a mixture of 1 μg of viral vector, 0.5 μg of packaging vector (pCL-eco or gag/pol for retrovirus, psPAX2 for lentivirus), 0.5 μg of coat protein (VSVG or pHCMV-EcoEnv), 5 μl of X-tremeGENE^TM 9 (Sigma), and 95 μl of Opti-MEM^TM (Gibco). The transfection mix was prepared using the manufacturer's protocol and added to the cells for 16–24 h, after which the mix was removed and the cells were fed with fresh full-growth medium. Cells were then cultured for an additional 24 h, after which the culture supernatant was collected and filtered through a 0.45-μm filter and then either stored at −80 °C or immediately used. For stable transduction, cells were plated at roughly 50% confluence, and then, 16–24 h later, viral supernatant diluted 1:1 with fresh growth medium was added to the cells. Polybrene (Sigma) was also added at 8 μg/ml. After 24 h, viral supernatants were removed, and cells were fed with fresh growth medium and then stably selected with the appropriate antibiotic.

Lamar J.M., Xiao Y., Norton E., Jiang Z.G., Gerhard G.M., Kooner S., Warren J.S, & Hynes R.O. (2018). SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis. The Journal of Biological Chemistry, 294(7), 2302-2317.

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C6 Glioma Cell NMDA Receptor Transfection

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Rat C6 glioma cells were gifted by Dr Nathalie Chevalier (INSERM U1198). They were maintained in DMEM supplemented by 10% fetal calf serum in the presence of 100 U/ml penicillin and 100 μg/ml streptomycin. C6 cells were transfected at 80% confluence with a combination of GluN1 and GluN2A or GluN2B (3.75 µg cDNA/wells) + 10 µl of Lipofectamine (Invitrogen) diluted in Opti-MEM^TM (Gibco^TM). The cells were used 24 h after transfection. All constructions of NMDA receptor subunits have been kindly provided by Pierre Paoletti (IBENS, ENS, Paris). C6 cells were used for less than 15 passages.

Sebih F., Mokrane N., Fontanel P., Kayatekin M., Kaabeche M., Guiramand J., Cohen-Solal C., Cens T., Rousset M., Charnet P., De Jésus Ferreira M.C., Thibaud J.B., Ménard C., Cantel S., Rolland V., Vignes M, & Roussel J. (2022). The Glutathione Metabolite γ-Glutamyl-Glutamate Partially Activates Glutamate NMDA Receptors in Central Neurons With Higher Efficacy for GluN2B-Containing Receptors. Frontiers in Pharmacology, 12, 794680.

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