Lipod293 transfection reagent

Manufactured by SignaGen

Sourced in United States

LipoD293 is a cationic lipid-based transfection reagent formulated for efficient DNA and RNA delivery into a variety of mammalian cell lines, including HEK293 cells. It facilitates the formation of lipid-nucleic acid complexes that can be easily taken up by cells.

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LipoD293 (77 citations) LipoD293™ In Vitro DNA Transfection Reagent (29 citations) LipoD293 reagent (13 citations) LipoD293™ DNA transfection reagent (4 citations) Transfection reagent (2 citations) LipoD293 In Vitro DNA Transfection Reagent Ver II (2 citations) LipoD293 in vitro transfection reagent (2 citations)

23 protocols using lipod293 transfection reagent

Mesenchymal Stem Cell Isolation and Manipulation

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MSC were isolated from 8-10 wk male C57BL/6 mice were prepared after Peister et al (Peister et al., 2004 ). For phosphorylation measurements, seeding density was 10,000 cells/cm² and 3000 cells/cm² for immunostaining experiments. All groups were cultured for 48h before beginning experiments and were serum starved overnight in serum free medium. For transiently silencing specific genes, cells were transfected with gene-specific small interfering RNA (siRNA) or control siRNA (20 nM) using PepMute Plus transfection reagent (SignaGen Labs, Rockville, MD) according to manufacturer’s instructions. PEGFP-C1-βcatenin (#16071) plasmid was purchased from Addgene. mdMSCs were transfected using 1μg DNA per 100,000 cells using LipoD293 transfection reagent (SignaGen Labs) according to manufacturer’s instructions. Please see Table S3 for specific siRNA sequences and Table S7 for reagent concentrations. Transfections and siRNA were applied 72h prior to Strain, LIV or SB415286. A complete list of all the reagents, DNA/RNAi sequences, antibodies and primers along with their final concentrations are provided in the supplementary appendix.

Uzer G., Bas G., Sen B., Xie Z., Birks S., Olcum M., McGrath C., Styner M, & Rubin J. (2018). Sun-mediated Mechanical LINC between Nucleus and Cytoskeleton Regulates βcatenin Nuclear Access. Journal of biomechanics, 74, 32-40.

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HEK293 Cell Culture and Transfection Protocol

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HEK293 cells were propagated at 37°C/5% CO₂ in DMEM growth medium supplemented with 10% FBS (Thermo Fisher Scientific). For imaging experiments, cells were plated on poly-D-lysine (PDL; MilliporeSigma) –coated coverglass in 24-well plates and transfected with 1.5 μg plasmid DNA per well using Xtreme HP transfection reagent (MilliporeSigma). For imaging experiments using LOV-based constructs, cells were imaged at 9–16 h after transfection, and for all other constructs cells were imaged 24 h after transfection. For NRG3 processing experiments (Figs. 3 C and S2 A), HEK293 cells were seeded in 12-well plates, transfected with 1 µg plasmid DNA per well using LipoD293 transfection reagent (SignaGen), and processed 24–48 h later for Western blotting. Rab transfection experiments were done by using 1.5 μg DNA of the respective plasmids per well of a 12-well plate using Xtreme HP transfection reagent (MilliporeSigma). Imaging was done after 16–20 h of transfection.

Ahmad T., Vullhorst D., Chaudhuri R., Guardia C.M., Chaudhary N., Karavanova I., Bonifacino J.S, & Buonanno A. (2022). Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3. The Journal of Cell Biology, 221(7), e202110167.

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Overexpression of Mutant NS2 Splice Variant

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The NS1 open reading frame containing a mutation in the splice site for NS2 was cloned into the pcDNA3.1 mammalian expression vector as previously described [14 (link),27 (link)]. Plasmids were transfected into U2OS cells for at least 16 h using the LipoD293 transfection reagent (SignaGen Laboratories, Frederick, MD, USA).

Larsen C.I, & Majumder K. (2023). The Autonomous Parvovirus Minute Virus of Mice Localizes to Cellular Sites of DNA Damage Using ATR Signaling. Viruses, 15(6), 1243.

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Inhibition of Salivary Tumor Cell Proliferation

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Exponentially growing salivary gland tumor cells were plated onto 96-well plates at the density of 1 × 10⁴ cells/well and transfected with either pCI blast (empty vector) or pCI blast-WIF1 using LipoD293 transfection reagent as per manufacturer's instruction (SignaGen Laboratories, Ijamsville, MD, USA). The transfection efficiency is ∼55% for all cell lines used in this study. Cell proliferation was assessed at different time intervals (24, 48 and 72 h) by hexosaminidase assay.^{73 (link)} The relative cell growth (proliferation) was determined by normalizing the cells with the respective control (i.e., vector) as we described previously.^{21 (link)}

Ramachandran I., Ganapathy V., Gillies E., Fonseca I., Sureban S.M., Houchen C.W., Reis A, & Queimado L. (2014). Wnt inhibitory factor 1 suppresses cancer stemness and induces cellular senescence. Cell Death & Disease, 5(5), e1246-.

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STAT5A and Akt Overexpression Protocols

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Retroviral-expressing vectors of wild-type STAT5A (pMSCV-STAT5A-GFP) and GFP-only (pMSCV-GFP) were provided by Dr. Hennighausen (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD, USA). Akt-IRES-GFP (active Akt) vector was the gift of Dr. Yukiko Gotoh (University of Tokyo).^{27 (link)} For making retrovirus, Plate-E cells were transfected with retroviral vectors and pEco (the plasmid containing packaging sequences) using LipoD293 transfection reagent (SignaGen Laboratories, Rockville, MD, USA). All supernatants were collected 48 h after transfection and passed through 0.45 μm filter, followed by 1000 g centrifugation. Retrovirus was added into BM culture at D2 (active Akt) or D3 (STAT5A) in the presence of 4 μg/ml polybrene (Sigma-Aldrich). Cells were harvested at D6 (active Akt) or D8 (STAT5A), washed with PBS, stained with anti-MHCII-APC, and then subjected to flow cytometry. GFP-positive and -negative cells were gated, respectively, and MHCII expression was analyzed.

So E.Y, & Ouchi T. (2014). Translational initiation regulated by ATM in dendritic cells development. Cell Death & Disease, 5(9), e1418-.

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Inducible Expression of Murine SphK2

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Doxycycline (DOX) inducible expression plasmid encoding SphK2 was generated by PCR from pVB201 (provided by Stephen Alexander, University of Missouri-Columbia) (Min et al., 2007 (link)) with primers 5’ -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT ACC ACC ATG GGG GGT TCT CAT CAT CAT- 3’ and 5’ -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG TCA GGC TTG TGG CTT TTG ACC TGC AGG- 3’. The amplified murine SphK2-encoding fragment was cloned into pINDUCER20 vector using BP and LR clonase kits (Invitrogen) according to the manufacturer’s instructions. The pINDUCER20 reagents were a gift from David Pintel at University of Missouri-Columbia (Adeyemi et al., 2014 (link); Meerbrey et al., 2011 (link)). For transient expression, HEK293 cells (2 00D7 10⁵/well) were seeded in a 24-well plate one day before transfection. Cells were then transfected with a plasmid encoding SphK2 (250 ng/well) using LipoD293 transfection reagent (SignaGen) and protocols recommended by the manufacturer. DOX (100 ng/ml, MP Biomedical) was added to the cell culture 24 hours post transfection to induce the transient expression of SphK2.

Xia C., Seo Y.J., Studstill C.J., Vijayan M., Wolf J.J, & Hahm B. (2018). Transient inhibition of sphingosine kinases confers protection to influenza A virus infected mice. Antiviral research, 158, 171-177.

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Transient Transfection of Mutant SOD1 in CHO Cells

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CHO cells were plated onto CaF₂ as previously described and, after 24–48 h, were transiently transfected with Lipo-D 293 transfection reagent (SignaGen) to overexpress mutant D125H SOD1-YFP protein. Cells were incubated for an additional 9 h post transfection under standard conditions prior to assembly of the flow cell. Fluorescent cells were identified and selected via light microscopy using a 10× objective. For this study, individual highly fluorescent cells were chosen for high aggregation potential and ease of distinguishing cytosolic and nuclear compartments when generating regions of interest (ROIs) for data processing.

Gelfand P., Smith R.J., Stavitski E., Borchelt D.R, & Miller L.M. (2015). Characterization of Protein Structural Changes in Living Cells Using Time-Lapsed FTIR Imaging. Analytical chemistry, 87(12), 6025-6031.

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Immunofluorescence Visualization of Viral Proteins

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HeLa cells (2.5 × 10⁵, ATCC) growing on the coverslips were transfected with each vector (0.5–1 μg) encoding a FLAG-tagged viral protein by using LipoD293 transfection reagent (SignaGen Laboratories). The cells at 24 h after transfection were fixed, permeabilized, and stained with a monoclonal anti-FLAG M2 antibody (Sigma-Aldrich, # F1804) in combination with Alexa Flour488-labeled anti-mouse secondary antibody (Thermo Fisher Scientific, #A11029) as described before [75 (link),76 (link)]. The cell nuclei were counterstained by Hoechst 33342 dye (Thermo Fisher Scientific, #H3570).

Xue X.Y., Majerciak V., Uberoi A., Kim B.H., Gotte D., Chen X., Cam M., Lambert P.F, & Zheng Z.M. (2017). The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues. PLoS Pathogens, 13(11), e1006715.

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Generating Raptor-Silencing Lentivirus

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Plasmid expressing raptor specific shRNA was made by cloning of double stranded oligonucleotide into the LKO.1 vector AgeI/EcoRI sites61 (link). The sequence of the oligonucleotide was:
Raptor_shRNA sense: CCGGGGCTAG TCTGTTTCGA AATTTCTCGA GAAATTTCGA AACAGACTAG CCTTTTTG.
Raptor_shRNA antisense: AATTCAAAAA GGCTAGTCTG TTTCGAAATT TCTCGAGAAA TTTCGAAACA GACTAGCC.
Plasmid expressing raptor specific shRNA was co-transfected with pCMV-dR8.2 DVPR vector and pCMV-VSV-G vector at the ratio of 4:3:1 using LipoD293 Transfection Reagent (SignaGen Laboratories, SL100668) into HEK293T cells to allow packaging of the lentivirus. Virus containing supernatants were collected at 24 h intervals for three consecutive days and were used as source of virus. HLFs were plated in 6-well plates and the lentivirus transduction was carried out in media containing 8 μg/ml hexadimethrine bromide (polybrene). Transduction efficiency was determined by monitoring the viral marker, RFP. The knockdown of raptor was confirmed by Western blot.

Zhang Y, & Stefanovic B. (2017). mTORC1 phosphorylates LARP6 to stimulate type I collagen expression. Scientific Reports, 7, 41173.

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Silencing Genes in Mouse Mesenchymal Stem Cells

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MSC were isolated from 8- to 10-week male C57BL/6 mice were prepared after Peister et al.^{94 (link)} For transiently silencing specific genes, cells were transfected with gene-specific small interfering RNA (siRNA) or control siRNA (20 nM) using PepMute Plus transfection reagent (SignaGen Labs, Rockville, MD) according to manufacturer’s instructions. MSCs were transfected using 1 µg DNA per 100,000 cells using LipoD293 transfection reagent (SignaGen Labs) according to manufacturer’s instructions. Transfections and siRNA were applied 72 h prior to sMG. A complete list of all the reagents (Table S2), antibodies (Table S3), siRNA sequences (Table S4), and primers (Table S5) along with their final concentrations are provided in the supplementary appendix.

Touchstone H., Bryd R., Loisate S., Thompson M., Kim S., Puranam K., Senthilnathan A.N., Pu X., Beard R., Rubin J., Alwood J., Oxford J.T, & Uzer G. (2019). Recovery of stem cell proliferation by low intensity vibration under simulated microgravity requires LINC complex. NPJ Microgravity, 5, 11.

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