Lipofecamine 2000

Manufactured by Thermo Fisher Scientific

Sourced in United States

Lipofecamine 2000 is a transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into mammalian cells. It is a cationic lipid-based formulation that forms complexes with nucleic acids, facilitating their uptake by the cells.

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9 protocols using lipofecamine 2000

Tetherin Knockdown in Corneal Epithelium

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Ambion Silencer Select siRNA (Invitrogen) was used for corneal knockdown experiments and transfection mediated using lipofectamine 2000 (Invitrogen) as described previously.^{8 (link)} Briefly, partial epithelial debridement of corneas of anesthetized WT mice was conducted to prepare the eye for transfection. Next, 3.33 µmol tetherin-specific or nonspecific scramble control siRNA in supplement-free DMEM media was applied to each cornea in a 10 µl eye drop containing 5 µl Lipofecamine 2000 (Invitrogen). siRNA sequences were designed and validated by the manufacturer. Mice were infected with HSV-1 16 hours post-transfection. On days 2, 3, and 5 pi, tissue was harvested for plaque assay and validation of knockdown efficiency. Tetherin transcript expression was quantified using PrimePCR technology (Biorad) as described for the gene array. Tetherin protein expression was imaged in immunolabeled cornea flat mounts by confocal microscopy to evaluate knockdown efficiency.

Royer D.J, & Carr D.J. (2015). A STING-dependent innate sensing pathway mediates resistance to corneal HSV-1 infection via upregulation of the antiviral effector tetherin. Mucosal immunology, 9(4), 1065-1075.

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Imaging Transfected Mouse Hepatocytes

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Primary cultured mouse hepatocytes were transfected using Lipofecamine 2000 (Invitrogen) and maintained in William's E Media prior to image analysis. Twenty-four hours post-transfection, hepatocytes were washed once with 1x PBS and subsequently stained with Hoechst 33432 for an additional 30 minutes. To visualize, mouse hepatocytes were washed three times with 1x PBS and then maintained in Opti-MEM® I Reduced Serum Media during fluorescence protein image analysis. Fluorescent proteins were imaged with a 30x air objective, and excited at either 400 nm (GFP) or 561 nm (RFP). The nuclei were visualized using Hoechst 33432 staining under ultraviolet light.

Cui W., Sun M., Zhang S., Shen X., Galeva N., Williams T.D, & Staudinger J.L. (2016). A SUMO-Acetyl Switch in PXR Biology. Biochimica et biophysica acta, 1859(9), 1170-1182.

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Lentiviral Vector Production and Quantification

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HIV-1 NL4-3, NLENY1, and YU-2 were packaged in 293T cells with native HIV-1 envelope or pseudotyped with VSV-G envelope. Viral packaging was done with 10–17 µg of each lentiviral construct and 3–4 µg of VSV-G plasmid DNA, using Lipofectamine plus or Lipofecamine-2000 (Invitrogen) as described earlier [7] (link),[18] (link),[59] (link). After 16 h of transfection, cells in 100-mm dishes were washed twice with fresh medium and incubated in RPMI 1640 containing 10% FBS. Culture supernatants were harvested 72–80 h after transfection, centrifuged at 1,200 rpm for 15 min, and filtered through a 0.20 µm filter. The filtrate, after the addition of MgCl2 (4 mM), was digested with 10–50 units of RNase-free DNase (Invitrogen) per 1 µg of plasmid DNA for 30 min at 37°C, then aliquoted and stored at −80°C. The viral stocks were quantified for p24 antigen by ELISA (ZeptoMetrix). A viral inoculum of 50–1,000 ng/mL was used to infect lymphocytes, HFA, and SVGA.

, & Chauhan A. (2014). Unperturbed Posttranscriptional Regulatory Rev Protein Function and HIV-1 Replication in Astrocytes. PLoS ONE, 9(9), e106910.

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Targeting circHIPK3 and miR-876-5p in Gastric Cancer

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Specific small interfering RNA (siRNA) against circHIPK3 (si-circHIPK3) and siRNA scrambled control (si-NC), and short hairpin RNA objecting circHIPK3 (sh-circHIPK3) and shRNA scrambled control (sh-NC) were purchased from GenePharma (Shanghai, China). The miR-876-5p mimic (miR-876-5p) and its negative control (miR-NC), and miR-876-5p inhibitor (anti-miR-876-5p) and its negative control (anti-miR-NC) were designed and acquired from Thermo Fisher Scientific (Waltham, MA, USA). HGC-27 and AGS cells were transfected with plasmids or oligonucleotides using Lipofecamine2000 (Invitrogen, Carlsbad, CA, USA). The sequences for si-circHIPK3, si-NC, sh-circHIPK3 and sh-NC were as follows: si-circHIPK3: 5′-CUACAGGUAUGGCCUCACA-3′, si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, sh-circHIPK3: 5′-ccggCUACAGGUAUGGCCUCACAttcaagagaTGTGAGGCCATACCTGTAGTTTTTTGGTACC-3′, sh-NC: 5′- ccggUUCUCCGAACGUGUCACGUTTttcaagagaAATCGTGACACGTTCGGAGAATTTTTTGGTACC-3′. For overexpression of PIK3R1 (PIK3R1), the primers were used for amplification and then cloned in the mammalian expression pcDNA3.1 vector (Invitrogen), PIK3R1 F-5′-CCGGAATTCATGAGTGCTGAGGGGTACCAGTAC-3′; and R-5′-CCGCTCGAGATCGCCTCTGCTGTGCATATATA-3′.

Li Q., Tian Y., Liang Y, & Li C. (2020). CircHIPK3/miR-876-5p/PIK3R1 axis regulates regulation proliferation, migration, invasion, and glutaminolysis in gastric cancer cells. Cancer Cell International, 20, 391.

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Transfection of Renal Cancer Cell Lines

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OS-RC-2 and 786-O human RCC cell lines were obtained from the Chinese Academy of Science (Shanghai, China) and cultured in Roswell Park Memorial Institute-1640 (RPMI 1640) (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovineserum (Gibco), 1% streptomycin/ penicillin, at 37 °C and 5% CO2. The OS-RC-2 and 786-O cell lines were cultured all night in a six-well plate (2.0 x 10⁵ cells per well) and then transfected with the pcDNA-MAN1C1 plasmid or control vector(Vigenebio, China) when cells were grown to 75%. Cells were transfected with Lipofecamine 2000 (Invitrogen, Carlsbad, CA, USA).

Li H., Wang G., Yu Y., Jian W., Zhang D., Wang Y., Wang T., Meng Y., Yuan C, & Zhang C. (2018). α-1,2-Mannosidase MAN1C1 Inhibits Proliferation and Invasion of Clear Cell Renal Cell Carcinoma. Journal of Cancer, 9(24), 4618-4626.

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Production and Quantification of HIV-1 Pseudotypes

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HIV-1 NL4-3 and NLENY1 were packaged in 293T cells either as wild-type or pseudotyped with VSV-G envelope. All plasmids were prepared endotoxin-free, using a maxiprep kit (Qiagen). Viral packaging was done with 17 μg of each HIV-1 infectious DNA clone alone or with 3-4 μg of VSV-G plasmid DNA, using Lipofecamine-2000 (Invitrogen) as described earlier [12 (link)]. After 16 h of transfection, cells in 100-mm dishes were washed twice with fresh medium and incubated in RPMI 1640 containing 10% FBS and 1% each streptomycin, penicillin, and amphotericin B (Invitrogen). The culture supernatants were harvested 72-80 h after transfection, centrifuged at 1,200 rpm for 15 min, and filtered through a 0.20 μm filter. The filtrate, after the addition of MgCl2 (4 mM), was digested with 10-50 units of RNase-free DNase (Invitrogen) per 1 μg of plasmid DNA for 30 min at 37°C, then aliquoted and stored at −80°C. The viral stocks were quantified for p24 antigen by ELISA (ZeptoMetrix). An NL4-3 viral inoculum of 1.0 μg/mL and VSV-NLENY1 of 100 ng/mL of p24 was used to infect HFA and SVGA.

, & Chauhan A. (2015). Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent. Microbes and infection / Institut Pasteur, 17(9), 651-659.

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Modulating cTFRC Expression in Bladder Cancer

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Short hairpin RNA (shRNA) of cTFRC were synthesized by GenePharma (Shanghai, China), shcTFRC targeting to the junction region of the cTFRC sequence. The shRNA plasmid of TFRC was purchased from Santa Cruz (Dallas, USA). EJ and T24 cells were transfected with cTFRC and TFRC shRNAs plasmids using Lipofecamine 2000 (Invitrogen, Carlsbad, CA, USA). The sequences of the effective shRNAs were provided in Additional file 1: Table S2. The full-length cTFRC cDNA was cloned into pCDH-CMV-MCS-EF1-GFP + Puro (Geneseed Biotech, Guangzhou, China) to obtain the pCDH-cTFRC overexpression of cTFRC. Production of lentiviral particles and transduction of BC cells was performed as described [14 (link)].

Su H., Tao T., Yang Z., Kang X., Zhang X., Kang D., Wu S, & Li C. (2019). Circular RNA cTFRC acts as the sponge of MicroRNA-107 to promote bladder carcinoma progression. Molecular Cancer, 18, 27.

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Lentiviral Vector Production and Quantification

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HIV-1 NL4-3, NLENY1, NLYUV3, YU-2, lentiviral pLVX-ZsGreen (Clontech), pLVX-Tat, and pLVX-Rev expression vectors were packaged in 293T cells with native HIV-1 envelope or pseudotyped with VSV-G envelope. Viral packaging was done by using 10–17 μg of each lentiviral construct and 3–4 μg of VSV-G plasmid DNA using Lipofectamine plus or Lipofecamine-2000 (Invitrogen) as described earlier (Mehla et al., 2010 (link); Vijaykumar et al., 2008 (link); Zhang et al., 2010 (link)). Lentiviral vectors were packaged using 12 μg pLVX vector, 8 μg gag-pol, 4 μg VSV-G, 3 μg Tat, 4 μg Rev, and 3 μg Vpr plasmids to achieve a workable titer. After 16 h of transfection, cells in 100 mm dishes were washed twice with fresh medium and incubated in RPMI 1640 containing 10% FBS. The culture supernatants were harvested 72–80 h after transfection, centrifuged at 1200 rpm for 15 min, and filtered through a 0.20 μm filter. The filtrate after addition of MgCl2 (4 mM) was digested with 10–50 units of RNase-free DNase (Invitrogen) per 1 μg of plasmid DNA for 30 min at 37°C, aliquoted and stored at −80°C. The viral stocks were quantified for p24 antigen by ELISA (ZeptoMetrix) or titrated for lentiviral particles on reporter cells. A viral inoculum of 50–1000 ng/mL was used for infection of Jurkat, primary macrophages, HFA, and SVGA.

Chauhan A., Mehla R., Vijayakumar T.S, & Handy I. (2014). Endocytosis-Mediated HIV-1 Entry and Its Significance in the Elusive Behavior of the Virus in Astrocytes. Virology, 0, 1-19.

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Murine MORC2 cDNA Overexpression in Lung Cancer

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Murine MORC2 cDNA (Origene, Rockville, MD, USA) was cloned into a pcDNA3.1 plasmid (pcDNA3.1-mMORC2). Lipofecamine 2000 (Invitrogen, Carlsbad, CA, USA) was then used to stably transfect LL/2 cells with pcDNA3.1-mMORC2 or vector. The isolated stable transfectants, LL/2-V and LL/2-MORC2, were isolated by antibiotic selection in media supplemented with 500 µg/ml Zeocin (Invitrogen, Carlsbad, CA, USA). PC-9 cells were transfected with lentiviral constructs tagged with green fluorescent protein (GFP) containing MORC2 shRNA (shMORC2) or scrambled control (shCon) and packaging plasmids (addgene, Cambridge, MA, USA) using Lipofectamine 2000. At 48 hours posttransfection, supernatants were gathered, and lung cancer cells were immediately infected in media containing 10 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA). Puromycin (5 μg/ml) was used as an antibiotic selection agent for cells containing MORC2 shRNA, and these cells were subsequently sorted for the top 10-20% of GFP-positive cells.

Liu M., Sun X, & Shi S. (2018). MORC2 Enhances Tumor Growth by Promoting Angiogenesis and Tumor-Associated Macrophage Recruitment via Wnt/β-Catenin in Lung Cancer. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 51(4).

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