Opti mem 1 reduced serum medium

Manufactured by Thermo Fisher Scientific

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Opti-MEM I Reduced Serum Medium is a cell culture medium formulated to support the growth and maintenance of a variety of cell types while reducing the amount of serum required. It is designed to optimize cell viability and transfection efficiency.

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Opti-MEM (8992 citations) Opti-MEM medium (2162 citations) Opti-MEM I (686 citations) MEM medium (359 citations) Opti-MEMTM (50 citations) OPTI-medium (29 citations) Reduced serum medium (10 citations) Opti-MEM I (1×) (7 citations) Opti-MEMTM I Reduced Serum Medium (7 citations)

876 protocols using opti mem 1 reduced serum medium

Silencing N-cadherin in Melanoma

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Silencer siRNAs targeting N-cadherin (siRNA ID: s2771) and scramble Silencer^® siRNA control were purchased from Thermo Fisher Scientific (Rochester, NY). Melanoma cells and CAFs were seeded in 6 cm dishes at an initial cell density of 1 × 10⁵ cells, cultured for 24 hours until 60–70% confluence, and then transfected with siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Rochester, NY). According to the manufacturer’s protocol, N-cadherin siRNA (10 μM) was diluted in 250 μl of Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Rochester, NY) and mixed with 15 μl of Lipofectamine RNAiMAX in 250 μl of Opti-MEM I reduced serum medium. After incubation at room temperature for 10 minutes, the mixture was added to the cells and incubated for three days. Afterward, the medium containing siRNA and RNAiMAX was replaced with regular DMEM culture medium.

Xio Y., Zhou L., Andl T, & Zhang Y. (2023). YAP1 controls the N-cadherin-mediated tumor-stroma interaction in melanoma progression. Research Square.

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UCP2 Overexpression in HK-2 Cells

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To upregulate the expression of UCP2 in HK-2 cells, UCP2-DNA transfection using lipofectamine 2000 was performed. Before treatment with AA I or AA I + genipin, HK-2 cells were incubated for 24 h with the complex UCP2 DNA-Lipofectamine 2000 in reduced serum medium (Opti-MEM I Reduced Serum Medium, Thermo Fisher Scientific). To be specific, 4 μg of UCP2-DNA (Sino bilogical, Beijing, China) were diluted into 200 μL of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Shanghai, China), and then 10 μL of lipofectamine 2000 were diluted in 200 μL of Opti-MEM I Medium, and then incubated for 5 min at room temperature. After 5 min of incubation, the diluted UCP2-DNA was combined with the diluted Lipofectamine 2000. After 20 min of incubation at room temperature, the 400 μL of DNA-Lipofectamine 2000 complexes were added to each well, and then the cells were incubated at 37 ℃ in a CO₂ incubator for 24 h for transgene expression.

Feng C., Anger E.E., Zhang X., Su S., Su C., Zhao S., Yu F, & Li J. (2022). Protective Effects of Mitochondrial Uncoupling Protein 2 against Aristolochic Acid I-Induced Toxicity in HK-2 Cells. International Journal of Molecular Sciences, 23(7), 3674.

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DCLK1 Knockdown in A549 Cells

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Briefly, 60 pmol DCLK1 siRNA (5′-GGGAGUGAGAACAAUCUACTT-3′ forward and 5′-UUCUCCGAACGUGUCACGUTT-3′ reverse) or negative control siRNA (Shanghai GenePharma Co., Ltd.) was diluted in 50 μl Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Inc.) without serum (the final concentration of RNA when added to the cells was 100 nM) and mixed gently. In addition, Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) was mixed gently before use, and then 1 μl Lipofectamine 2000 was diluted in 50 μl Opti- MEM I Reduced Serum Medium. It was then mixed gently and incubated for 5 min at room temperature. Next, the diluted oligomer was combined with the diluted Lipofectamine 2000, mixed gently and incubated for 20 min at room temperature. The oligomer-Lipofectamine 2000 complexes were added to 30–50% confluent A549 cells with 500 μl complete medium, followed by gentle mixing by rocking the plate back and forth. The cells were incubated at 37°C in a CO₂ incubator for 48 h before the gene knockdown assay.

Yang W.Q., Zhao W.J., Zhu L.L., Xu S.J., Zhang X.L., Liang Y., Ding X.F., Kiselyov A, & Chen G. (2021). XMD-17-51 Inhibits DCLK1 Kinase and Prevents Lung Cancer Progression. Frontiers in Pharmacology, 12, 603453.

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Transfection of Cells with siRNA and Plasmids

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Transfection of cells with siRNA was performed via lipofection using Lipofectamine 2000 or Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s protocol and as described in Zimmer et al. (2011) [81 (link)]. Mouse Dnmt1 siRNA (30 nM; #sc-35203, Santa Cruz Biotechnology, Dallas, TX, USA) or control siRNA (15 nM; Block-iT Alexa Fluor red (#14750100) or Block-iT green (#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) were applied for 5 h in antibiotic- and serum-free Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and cells were then grown in respective cell culture media. For co-transfection we applied 30 nM Dnmt1 siRNA (#sc-35203, Santa Cruz Biotechnology), 15 nM control siRNA (Block-iT Alexa Fluor red (#14750100) or Block-iT green ((#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) and plasmid DNA (pLAMP1-mCherry, 200 ng/µL; CD63-pEGFP C2, 200 ng/µL; 1× GFP-pEGFP N3-HTT, 260 ng/µL) with Lipofectamine 2000 (Thermo Fisher Scientific), as described in the protocol of the manufacturer. Antibiotic- and serum-free Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) were used for preparation of transfection reagents and dilutions. Co-transfections were performed for 24 h prior to live cell imaging.

Bayer C., Pitschelatow G., Hannemann N., Linde J., Reichard J., Pensold D, & Zimmer-Bensch G. (2020). DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes. International Journal of Molecular Sciences, 21(15), 5420.

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RIOK2 Knockdown by siRNA Transfection

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Lipofectamine 2000 (Invitrogen) was used for siRNA transfection according to the manufacturer’s instructions. Briefly, 50 µmol siRNA was diluted in 250 µL Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific), and 5 µL Lipofectamine 2000 was diluted in 250 µL Opti-MEM I Reduced Serum Medium. The diluted Lipofectamine 2000 was mixed with the diluted siRNA, and then the Lipofectamine/siRNA complex was applied to cell lines. These cells were seeded into 6-well plates (5.0 × 10⁵ cells per well) before incubation for 48 h. We purchased siRNA specific to RIOK2, constructed by QIAGEN (QIAGEN, Venlo, The Netherlands): RIOK2-siRNA_1 (5′-ATGAAACGTTTCAGCTACGAA-3′) and RIOK2-siRNA_2 (5′-TAGGAAGAACCTCGTTTCGAA-3′). As negative control siRNA, we used AllStars Negative Control siRNA (QIAGEN, Venlo, The Netherlands). Cells were harvested 48 h after transfection and used in further analysis.

Matsuzaki Y., Naito Y., Miura N., Mori T., Watabe Y., Yoshimoto S., Shibahara T., Takano M, & Honda K. (2022). RIOK2 Contributes to Cell Growth and Protein Synthesis in Human Oral Squamous Cell Carcinoma. Current Oncology, 30(1), 381-391.

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Transfecting Neurons with Fluorescent Plasmids

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The induced neurons at PID 45 were transfected with the plasmid DNA of pCl-DsRed (Supplementary Fig. 2A) and β-actin-EGFP [24 (link)]. Solution 1 (50 μL Opti-MEM™ I Reduced-Serum Medium (Gibco 31985-070) and 1 μL Lipofectamine® 3000 Reagent (ThermoFisher Scientific L3000001)) and solution 2 (50 μL Opti-MEM™ I Reduced-Serum Medium, 2 μL P3000™ Reagent (ThermoFisher Scientific L3000001), and a total of 1 μg plasmid DNA (0.5 μg pCl-DsRed and 0.5 μg β-actin-EGFP)) was mixed and incubated at room temperature for 5 min. The mixture was then added directly to the wells, and the plate was incubated at 37 °C for 1 h. After 1 h of incubation, the culture medium was replaced with the fresh neuronal differentiation medium supplemented with E2 or DMSO. Cells were fixed with 4 % PFA after 4 days of exposure to the E2 or DMSO solution. After immuno-staining of the cells by an anti-RFP antibody or anti-GFP antibody (Supplementary Table 2), cells were imaged using a fluorescence microscope (BZ-X810; Keyence, Osaka, Japan) and a confocal microscope (Zeiss LSM700; ZEISS Group, Oberkochen, Germany). Neurite complexity was analyzed by the protocol for Sholl analysis in the ImageJ software [25 (link)]. The number of synapses per unit length was analyzed using SynD software [26 (link)].

Supakul S., Oyama C., Hatakeyama Y., Maeda S, & Okano H. (2024). Estradiol enhanced neuronal plasticity and ameliorated astrogliosis in human iPSC-derived neural models. Regenerative Therapy, 25, 250-263.

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siRNA Knockdown of VCP Protein

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A pool of three siRNAs (MISSION; Sigma-Aldrich) targeted to VCP were used which had been designed using an algorithm from Rosetta Inpharmatics LLC (Washington, USA) and targeted the VCP gene at nucleotides 768 (Hs01_00118726), 1908 (Hs02_00343009) and 2548 (Hs01_00118728). In brief, MISSION VCP siRNA or a negative control scrambled siRNA (ThermoFisher Scientific) was diluted in Opti-MEM® I reduced serum medium (Life Technologies) at a final concentration of 100 nM. Lipofectamine RNAiMAX (ThermoFisher Scientific) at RT was briefly vortexed and added to the diluted siRNA at 6 μl/ml Opti-MEM® I reduced serum medium prior to vortexing and incubating at RT for 25 minutes to facilitate siRNA-lipid complex formation. Medium from cells was replaced with the transfection mix at 1 ml/well in 6-well plates and 250 μl/well in 24-well plates and gently mixed. 6 hours later, transfection mix was replaced with complete medium. Downstream analyses were performed at least 48 hours post-transfection.

Read M.L., Brookes K., Thornton C.E., Fletcher A., Nieto H.R., Alshahrani M., Khan R., Borges de Souza P., Zha L., Webster J.R., Alderwick L.J., Campbell M.J., Boelaert K., Smith V.E, & McCabe C.J. (2022). Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter. Cell Chemical Biology, 29(3), 502-516.e7.

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siRNA Knockdown and Rescue Assay for APE2

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For siRNA experiments, APEX2 siRNA (Dharmacon-HorizonDiscovery ON-TARGETplus Human APE2 siRNA Cat#L-013730-01-0005) or control siRNA (Dharmacon-HorizonDiscovery ON-TARGETplus non-targeting siRNA Cat#D-001810-01-05) was mixed with Lipofectamine^R RNAiMAX (Thermo Fisher Scientific Cat#13778100) in Opti-MEM I Reduced Serum Medium (Gibco Cat#31985070) and incubated for 3–5 days according to the manufacture’s protocol. The target sequences of the Dharmacon APE2 siRNA include 5′-GAGCCAUGUGAUGCGUA-3′, 5′-CAACAAUCAAACCCGGGUA-3′, 5′-GGACGAGCUGGAUG CGGAU-3′, and 5′-GAGAAGGAGUUACGGACCU-3′, whereas the non-targeting siRNA sequence is 5′- UGGUUUACAUGUCGACUAA-3′. For the rescue experiments in Figure 1B and Supplementary Figure 1A, after siRNA-mediated APE2-KD, transfecting control plasmid pcDNA3-YFP (Addgene Cat#13033) or pcDNA3-YFP-xAPE2 with Lipofectamine 2000 (Thermo Fisher Scientific Cat#116680019) in Opti-MEM I Reduced Serum Medium. After different treatment and incubation, cells were imaged via fluorescence microscopy to ensure YFP or YFP-xAPE2 was expressed in cells.

Hossain M.A., Lin Y., Driscoll G., Li J., McMahon A., Matos J., Zhao H., Tsuchimoto D., Nakabeppu Y., Zhao J, & Yan S. (2021). APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells. Frontiers in Cell and Developmental Biology, 9, 738502.

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HIF1A Knockdown and DFO Treatment

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Cells were plated onto 35-mm dishes at 3.0 × 10⁵ cells/dish and incubated for 12 h. Then, 1 μL of small interfering RNA (siRNA) targeting human HIF1A (MISSION® siRNA; Sigma-Aldrich; Merck KGaA) was diluted in 50 μL Opti-MEM® I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) and mixed with 3.5 μL of Lipofectamine® RNAiMAX Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) pre-diluted in 50 μL of Opti-MEM® I Reduced Serum Medium. After 20 min of incubation at room temperature, the complexes were added to the cells in a final volume of 1 mL of medium and cells were incubated for 24 h. As a control for siRNA, BLOCK-iT™ Alexa Fluor® Red Fluorescent Oligo 20 μM (Invitrogen; Thermo Fisher Scientific, Inc.) was used. After incubation for 24 h, cells were washed with Dulbecco’s phosphate-buffered saline (FUJIFILM Wako Pure Chemical), dissociated by trypsinization and plated onto new 35-mm dishes at 3.0 × 10⁵ cells/dish. After 24 h, 5 μM DFO was added to the culture medium and cells were incubated for 24 h.

Harada T., Hirose K., Wada Y., Sato M., Ichise K., Aoki M., Kato T., Takeda K, & Takai Y. (2020). YC-1 sensitizes the antitumor effects of boron neutron capture therapy in hypoxic tumor cells. Journal of Radiation Research, 61(4), 524-534.

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Lentiviral Transduction and Plasmid Transfection

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Following a 48 h transduction with lentiviruses as described above, cells were transfected with mEmerald-Lamin A-C-18 (addgene plasmid # 54138) and pEGFP-C2 (Clontech) by initially combining plasmid DNA with the Lipofectamine^™ 2000 Transfection Reagent (Invitrogen, Cat#11668019) in Opti-MEM^™ I Reduced Serum Medium (Gibco, Cat#31985070) for 20 minutes, and diluting the mix in cell media. The culture media of transduced cells were replaced with the plasmid DNA-containing media for 6 h, after which there was a change and overnight incubation with fresh media. Subsequently, cells were dissociated and plated on gelatin-coated coverslips as described below. All other transient transfections with shRNA-encoding plasmids were performed by combining plasmid DNA with the Lipofectamine^™ 2000 Transfection Reagent (Invitrogen, Cat# 11668019) in Opti-MEM^™ I Reduced Serum Medium (Gibco, Cat# 31985070) for 20 minutes, and adding the mix dropwise into cell culture media. Following 4–6 h incubation, transfected cell cultures were replaced with fresh complete growth medium and allowed to incubate for up to 72 h before seeding on matrix-coated coverslips for immunostaining.

Ha H.C., Juluri K., Zhou Y., Leung S., Hermankova M, & Snyder S.H. (2001). Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration. Proceedings of the National Academy of Sciences of the United States of America, 98(6), 3364-3368.

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