Lipofectamine 2000

Manufactured by RiboBio

Sourced in China

Lipofectamine 2000 is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as plasmids, siRNA, and mRNA, into eukaryotic cells. It forms complexes with the nucleic acids, which can then be taken up by the cells, facilitating the introduction of the genetic material.

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Lab products found in correlation

73 protocols using lipofectamine 2000

Prolinedehydrogenase Gene Knockdown and Overexpression

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Cells were transfected either with PRODH siRNA (100 nM) or non-targeting pool (control siRNA) using lipofectamine 2000 according to the manufacturer’s instructions (Ribo Bio Co., Ltd). After 48 h, the media was collected and then used for the indicated assays. PRODH (designated as siPRODH, target sequences were GATGCAGCGGAAGTTCAAT), and StealthTM RNAi negative controls (designated as siNEG) were purchased from RiboBio.
pcDNA 3.1 expression vectors encoding mouse PRODH were transfected into RM-1 using lipofectamine 2000 reagent according to the manufacturer’s instructions.
The efficiency of the transfection was determined by RT–PCR assay. After 48 h, the media was collected and then used for the indicated assays.

Yan Y., Chang L., Tian H., Wang L., Zhang Y., Yang T., Li G., Hu W., Shah K., Chen G, & Guo Y. (2018). 1-Pyrroline-5-carboxylate released by prostate Cancer cell inhibit T cell proliferation and function by targeting SHP1/cytochrome c oxidoreductase/ROS Axis. Journal for Immunotherapy of Cancer, 6, 148.

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Modulating miR-494 and SOX9 in NP cells

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MiR-494 mimic, miR-494 inhibitor, and their negative control (miR-Scr) were designed and synthesized by RiboBio (Guangzhou, China) and transfected into NP cells grown to 80% confluence using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For SOX9 knockdown, short interfering (si)RNA against SOX9 (siSOX9) and scrambled siRNA (siScr) (RiboBio) were co-transfected with miR-494 inhibitor or miR-Scr into NP cells using Lipofectamine 2000. After 48 h, cells were harvested and used for analyses.

Kang L., Yang C., Song Y., Zhao K., Liu W., Hua W., Wang K., Tu J., Li S., Yin H, & Zhang Y. (2017). MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells. Oncotarget, 8(17), 27868-27881.

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Silencing GPNMB in U87 Glioblastoma Cells

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U-87MG (U87) glioblastoma cell line of unknown origin was obtained from the American Type Culture Collection (cat. no. HTB-14). U87 cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Small interfering RNA (siRNA) targeting human GPNMB (5′-GGATAATACTGGCCTGTTT-3′) and a negative control siRNA (5′-GGCTCTAGAAAAGCCTATGC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. A total of 5×10⁵ U87 cells were seeded in 6-well plates overnight and then transfected with 50 nM siRNA using Lipofectamine^® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h according to the manufacturer's instructions. Subsequent experiments were conducted 24 h post-transfection.

Feng X., Zhang L., Ke S., Liu T., Hao L., Zhao P., Tu W, & Cang S. (2020). High expression of GPNMB indicates an unfavorable prognosis in glioma: Combination of data from the GEO and CGGA databases and validation in tissue microarray. Oncology Letters, 20(3), 2356-2368.

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siRNA-Mediated Knockdown of SND1

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For siRNA-mediated knockdown, cells were transfected with 10 nM of SND1-specific siRNAs or a scrambled siRNA sequence (siCtrl) as a control (purchased from Ribobio (Guangzhou, China), see Supplementary Table 1) using Lipofectamine 2000 on the day of plating and subjected to subsequent analysis.

Chen B., Wang M., Qiu J., Liao K., Zhang W., Lv Q., Ma C., Qian Z., Shi Z., Liang R., Lin Y., Ye J., Qiu Y, & Lin Y. (2022). Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling. Journal of Experimental & Clinical Cancer Research : CR, 41, 209.

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Silencing SNHG9 and miR-214-5p Regulation

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Short hairpin (sh)RNAs synthesized against SNHG9 (sh-SNHG9) and matched negative control sequences (sh-NC) were subcloned into the pGPU6/GFP/Neo vector (Shanghai GenePharma Co., Ltd.). Subsequently, SKOV3 cells were transfected with 100 pmol sh-SNHG9 or sh-NC using Lipofectamine^® 2000 (Thermo Fisher Scientific Inc.), according to the manufacturer's protocol. Stable cell clones were selected using 0.5 mg/ml of G418 (Sigma-Aldrich; Merck KGaA). Thereafter, the cells were transfected with 0.1 nmol miR-214-5p mimics, negative control (NC) mimics (miR-NC), miR-214-5p inhibitors (anti-miR-214-5p) or anti-miR-NC (Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000, according to the manufacturer's protocol. The sequences of miR-214-5p mimics are as follows: Sense, 5′-UGCCUGUCUACACUUGCUGUGC-3′ and antisense, 5′-ACAGCAAGUGUAGACAGGCAUU-3′. The sequences of miR-214-5p inhibitor are as follows: Sense, 5′-GCACAGCAAGUGUAGACAGGCA-3′. Subsequent experimentation was performed 48 h post-transfection.

Chen G.Y., Zhang Z.S., Chen Y, & Li Y. (2020). Long non-coding RNA SNHG9 inhibits ovarian cancer progression by sponging microRNA-214-5p. Oncology Letters, 21(2), 80.

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Regulation of TP73-AS1 and miR-654-3p in HAECs

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Small interfering RNA targeting TP73-AS1 (si-TP73-AS1, sense 5ʹ-CCCAGUGGUGACUCCACAATT-3ʹ and antisense: 5ʹ-UGGGUUAGGCCCCACCU GGTT-3ʹ) and corresponding negative control (si-NC, 5ʹ-ATTGGAAGCTGTGTTCCA TTA-3ʹ) were synthesized by Jima Biotech (Shanghai, China) and transfected into HAECs using Lipofectamine 2000 (Invitrogen, Carlsbad, California, U.S.A.). MiR-654-3p mimic, miR-NC, miR-654-3p inhibitor, inhibitor NC, TP73-AS1 overexpressing vector pc-TP73-AS1, AKT3 overexpressing vector pc-AKT3 and negative control vector pc-NC were purchased from RiboBio (Guangzhou, China) and transfected into HAECs using Lipofectamine 2000 following the manufacturer’s instructions.

Ni J., Huang Z, & Wang D. (2021). LncRNA TP73-AS1 promotes oxidized low-density lipoprotein-induced apoptosis of endothelial cells in atherosclerosis by targeting the miR-654-3p/AKT3 axis. Cellular & Molecular Biology Letters, 26, 27.

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Cholangiocarcinoma Cell Line Manipulation

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The human cholangiocarcinoma cell lines QBC939, RBEandHucct-1 obtained from Cell Bank of Type Culture of the Chinese Academy of Sciences (Shanghai, China) were cultured with RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10%FBS and penicillin/streptomycin at 37°C with 5% CO₂. After entering the logarithmic growth phase, cells were washed 3 times with PBS, digested with trypsin and planted into a 6-well plate. When the cell density in the well plate reached 50–60% confluence, the cells were transfected with siRNAs (50nM) using Lipofectamine 2000 (Invitrogen, USA). The siRNA targeting USP8 (si-USP8) was synthesized by OriGene (Beijing, China), and siRNA Negative Control (OriGene) was used as negative control (si-NC). The siRNA-USP8 sequences were 5ʹ-CCAAAGAGAAAGGAGCAAT-3ʹ (529–548). The pcDNA3.1-USP8 expressing vector was obtained from Ribobio (Guangzhou, China) and transfected into Hucct-1 cells using Lipofectamine 2000. IGF-1 was obtained from Abcam (Cambridge, UK) and cells were treated with IGF-1 (200 ng/mL) for 24 h.

Jing X., Chen Y., Chen Y., Shi G., Lv S., Cheng N., Feng C., Xin Z., Zhang L, & Wu J. (2020). Down-regulation of USP8 Inhibits Cholangiocarcinoma Cell Proliferation and Invasion. Cancer Management and Research, 12, 2185-2194.

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Circ_0058063 Overexpression in KYSE510 Cells

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KYSE510 cells were harvested at confluence of 70–80% to perform cell transfections. Vectors expressing Circ_0058063 and GLUT1 were constructed by RIBOBIO (Guangzhou, China). Circ_0058063 siRNA and negative control (NC) siRNA were also from RIBOBIO. All transfections were performed using lipofectamine 2000 (RIBOBIO) to transfect 40 nM siRNA (NC siRNA as NC group) and 10 nM vector (empty vector as NC group) into 10⁶ cells. Control (C) cells for all transfections were untransfected cells. Following studies were performed at 24 h post-transfection.

Zheng Y., Chen Y., Jiang H., Zhang H., Wang H., Xu J, & Yu Z. (2020). Circ_0058063 upregulates GLUT1 expression and promotes glucose-uptake in esophageal squamous-cell carcinomas. Journal of Thoracic Disease, 12(3), 925-931.

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Functional Analysis of miR-652-3p in Hepatocellular Carcinoma

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Human normal hepatocytes cell HL-7702 and HCC cell lines Huh-7, Hep3B, and HepG2 used in this study were all from the Chinese Academy of Sciences Cell Bank. Cells were cultured in a DMEM medium containing 10% FBS, 1% penicillin, and streptomycin in a moist incubator at 37°C, 5% CO₂. When the cells grow to the growth phase, miR-652-3p mimic (miR10022709-1-5, Ribo Bio, Guangzhou, China), mimic NC (miR1N0000001-1-5, Ribo Bio, Guangzhou, China), miR-652-3p inhibitor (miR2171122085637-1-5, Ribo Bio, Guangzhou, China), inhibitor NC (miR2N0000001-1-5, Ribo Bio, Guangzhou, China) are transferred into the cells used Lipofectamine 2000 for in vitro cell function detection of miR-652-3p.

Chi X., Jiang Y., Chen Y., Lv L., Chen J., Yang F., Zhang X., Pan F, & Cai Q. (2021). Upregulation of microRNA miR-652-3p is a prognostic risk factor for hepatocellular carcinoma and regulates cell proliferation, migration, and invasion. Bioengineered, 12(1), 7519-7528.

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Overexpression and Knockdown Protocols for HBX and Stress Signaling Genes

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For transient overexpression, an HBX DNA fragment was inserted into the pEGFP plasmids; the resulting plasmids were named as pEGFP-HBX. HepaRG cells were transfected with either pEGFP or pEGFP-HBX plasmids using Lipofectamine^® 2000 according to the manufacturer’s direction. For transient knockdown, cells were transfected with 50 nmol/L annealed double-stranded siRNA for PPP2R5C (si2R5C), HSPA5 (siHSPA5), CREBH (siCREBH) (Ribobio, Guangzhou, China) using Lipofectamine^®2000 according to the manufacturer’s mannual. siNC stands for siRNA negative control. After 24–72 h, cells were analyzed for their suppression efficiency using qPCR and western blot and used in other experiments. All experiments were repeated at least three times independently.

He C., Qiu Y., Han P., Chen Y., Zhang L., Yuan Q., Zhang T., Cheng T., Yuan L., Huang C., Zhang S., Yin Z., Peng X.E., Liang D., Lin X., Lin Y., Lin Z, & Xia N. (2018). ER stress regulating protein phosphatase 2A-B56γ, targeted by hepatitis B virus X protein, induces cell cycle arrest and apoptosis of hepatocytes. Cell Death & Disease, 9(7), 762.

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