Dotap liposomal transfection reagent

Manufactured by Merck Group

Sourced in Germany, United States

DOTAP Liposomal Transfection Reagent is a cationic liposome-based transfection reagent. Its core function is to facilitate the delivery of nucleic acids, such as DNA or RNA, into eukaryotic cells.

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DOTAP transfection reagent (3 citations) DOTAP (32 citations)

16 protocols using dotap liposomal transfection reagent

Amyloid-beta Induced Oxidative Stress

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Aβ peptide (Aβ_1-42) was from Bachem Inc. (Torrance, CA, USA). Fluo4-AM, MitoSOX^TM Red Mitochondrial Superoxide Indicator, MitoTracker^® Orange CMTMRos, anti-rabbit Alexa Fluor^® 488 and anti-mouse Alexa Fluor^® 635 were from Molecular Probes, Inc. (Eugene, OR, USA). Hexafluoro-2-propanol (HFIP) and CMC were from Merck (Darmstadt, Germany), Neurobasal and Dulbecco’s modified essential medium (DMEM), B27 supplement and lipofectamine 2000 were from Gibco (Carlsbad, CA, USA). DOTAP Liposomal Transfection Reagent was from Sigma–Aldrich (Oakville, ON, Canada). Phosphodiester oligonucleotides (ODNs) were from Integrated DNA Technologies (Coralville, IA, USA). The mito-Pericam plasmid was donated by Dr. V. Eisner. Bicinchoninic acid assay (BCA) kit and mHsp-70 antibody were from Pierce Biotechnology (Rockford, IL, USA). Ryanodine was from Alexis (Lausen, Switzerland). PDVF membranes were from Millipore (Bedford, MA, USA). RyR2 antibody and Rhod2-AM was from Thermo-Fisher (Waltham, MA, USA). Gp91 ds-tat was from AnaSpec (Fremont, CA, USA).

SanMartín C.D., Veloso P., Adasme T., Lobos P., Bruna B., Galaz J., García A., Hartel S., Hidalgo C, & Paula-Lima A.C. (2017). RyR2-Mediated Ca2+ Release and Mitochondrial ROS Generation Partake in the Synaptic Dysfunction Caused by Amyloid β Peptide Oligomers. Frontiers in Molecular Neuroscience, 10, 115.

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Bacterial DNA/RNA Isolation and Transfection

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Bacterial DNA and RNA isolation was performed as previously described [14 (link)]. Cells were transfected with 1 μg of RNA or DNA complexed or not with DOTAP liposomal transfection reagent (Sigma-Aldrich) as previously described [14 (link)].

Lavagna A., Auger J.P., Dumesnil A., Roy D., Girardin S.E., Gisch N., Segura M, & Gottschalk M. (2019). Interleukin-1 signaling induced by Streptococcus suis serotype 2 is strain-dependent and contributes to bacterial clearance and inflammation during systemic disease in a mouse model of infection. Veterinary Research, 50, 52.

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Characterization of N6-modified Adenosine Agonists

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The nineteen test agonists, N⁶-modified adenosine analogues, were synthesized and initially characterized pharmacologically at the hA₃AR overexpressed in CHO cells, as previously reported [15 (link)–17 (link)]. Reference agonists 2-Cl-IB-MECA (2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcar-boxamidoadenosine) and NECA (5′-N-ethylcarboxamidoadenosine) were purchased from Tocris Bioscience (Biotechne, Abingdon, UK). Poly-D-lysine hydrobromide and DOTAP Liposomal Transfection Reagent were purchased from Sigma-Aldrich (Steinheim, Germany). FuGene® HD and Nano-Glo Live Cell Reagent were provided by Promega (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with GlutaMAX®, Hank’s Balanced Salt Solution (HBSS), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), pertussis toxin (PTX), penicillin/streptomycin (10,000 IU/mL and 10,000 μg/mL) and amphotericin B (250 μg/mL) were bought from Thermo Fisher Scientific (Pittsburg, PA, USA). The anti-dNGFR (truncated nerve growth factor receptor) antibody was purchased from Chromaprobe (Maryland Heights, MO, USA). Human Embryonic Kidney (HEK) 293T cells (passage 20) were a kind gift of Prof. O. De Wever of the Laboratory of Experimental Cancer Research (Ghent University Hospital, Belgium). The cell lines CB₁-NanoBiT®-βarr2 and CB₁-NanoBiT®-miniGα_i were described before [14 ,18 (link),19 (link)].

Pottie E., Tosh D.K., Gao Z.G., Jacobson K.A, & Stove C.P. (2020). Assessment of biased agonism at the A3 adenosine receptor using β-arrestin and miniGαi recruitment assays. Biochemical pharmacology, 177, 113934.

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Stimulation of Dendritic Cells with Bacterial Agents

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Stimulation of the DCs was performed by adding RPMI plus 10% FBS (0.5 mL/well) containing B. abortus [multiplicity of infection (MOI) 100:1], Escherichia coli LPS (1 µg/mL; Sigma-Aldrich), or B. abortus total RNA (2 µg/mL) complexed with DOTAP Liposomal Transfection Reagent (Sigma-Aldrich, St. Louis, MO, USA). Briefly, DOTAP was mixed with bacterial RNA (5:1 ratio) in 100 µL/well serum-free RPMI and incubated for 20 min at room temperature. Then, complexes were added to the cells in the presence of 12.5% FBS and cell cultures were incubated for 24 h at 37°C in a 5% CO₂ atmosphere. Culture supernatants were collected after 24 h of stimulation and kept at −80°C until use. To the remaining cells, it was added 0.350 mL/well of TRIzol^® to further RNA isolation.

Campos P.C., Gomes M.T., Guimarães E.S., Guimarães G, & Oliveira S.C. (2017). TLR7 and TLR3 Sense Brucella abortus RNA to Induce Proinflammatory Cytokine Production but They Are Dispensable for Host Control of Infection. Frontiers in Immunology, 8, 28.

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Synthetic RNA Delivery to Endosomes

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To deliver RNAs to endosomes, we used DOTAP liposomal transfection reagent (Sigma-Aldrich) as previously described [11 (link),56 (link)]. In brief, 230 pmol or other various amounts of synthetic RNAs were mixed with 60 μl of HBS buffer and 15 μl of DOTAP reagent and incubated for 15 min. The RNA-DOTAP solution was then added to 1 ml HMDM or PHMDM medium, followed by incubation of the cells for 16 h.

Pawar K., Shigematsu M., Sharbati S, & Kirino Y. (2020). Infection-induced 5′-half molecules of tRNAHisGUG activate Toll-like receptor 7. PLoS Biology, 18(12), e3000982.

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Retroviral Transduction of Activated B Cells

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To produce retrovirus, pSIREN- or pMXs-based plasmids were cotransfected together with pVSVG into Plat-E cells (kindly provided by T. Kitamura, University of Tokyo) using PEI Max (Mw 40,000, 24765-1; Polysciences). The virus-containing supernatant was harvested 2 days after transfection. For retroviral transduction, B cells were preactivated in vivo: B1-8^hi mice were injected i.p. with 50 µg of NP-Ficoll, and then B cells were purified from the spleens of these mice on the next day. These B cells were mixed with the virus-containing supernatant and spin infected at 2000 rpm, 37°C for 90 min with 10 mg/ml DOTAP Liposomal Transfection Reagent (11202375001; Sigma). One day later, the cells were harvested and 5 × 10⁵ cells were transferred into B6 mice that had been immunized i.v. with NP-Ficoll on the previous day. This strategy is summarized in Figure 4—figure supplement 1B.

Fukao S., Haniuda K., Tamaki H, & Kitamura D. (2021). Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria. eLife, 10, e72116.

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Exosome Transfection and Cytokine Assays

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Morphine was purchased from R&D Systems (Minneapolis, MN, USA). Chemical inhibitors including the opioid receptor antagonist naltrexone, endosomal TLR inhibiter Chloroquin, IKK‐2 inhibitor SC514 were purchased from Sigma‐Aldrich (St. Louis, MO, USA). DOTAP Liposomal Transfection Reagent was also purchased from Sigma‐Aldrich. The mouse IL‐6 / TNFα DuoSet kits were obtained from R&D Systems (Minneapolis, MN, USA). Exo‐Fect^TM Exosome Transfection Kit was purchased from SBI System Bioscience (Palo Alto, Canada).

Liao K., Niu F., Hu G., Yang L., Dallon B., Villarreal D, & Buch S. (2020). Morphine‐mediated release of miR‐138 in astrocyte‐derived extracellular vesicles promotes microglial activation. Journal of Extracellular Vesicles, 10(1), e12027.

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Inflammasome Activation by Bacterial Factors

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Flagellin purified from P. aeruginosa was purchased from InvivoGen. DOTAP liposomal transfection reagent was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS), ATP, nigericin, pyocyanin, and proteinase K were purchased from Sigma-Aldrich. N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) were obtained from Cayman. Antibodies were acquired to detect mouse caspase-1 (Adipogen, 20B-0042), mouse IL-1β (R&D, AF-401-NA), mouse IL-6 (Cell Signaling, 12912), mouse NLRP3 (Adipogen, 20B-0014), mouse ASC (Santa Cruz, SC-22514-R), and Flagellin (InvivoGen, mabg-flapa).

Yang J., Lee K.M., Park S., Cho Y., Lee E., Park J.H., Shin O.S., Son J., Yoon S.S, & Yu J.W. (2017). Bacterial Secretant from Pseudomonas aeruginosa Dampens Inflammasome Activation in a Quorum Sensing-Dependent Manner. Frontiers in Immunology, 8, 333.

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Liver Cell Culture and Transfection

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HepG2.2.15 cells, HepG2 cells, and Huh7 cells were cultured at 37 °C in a CO₂ incubator. HepG2.2.15 cells were routinely cultured in RPMI-1640 medium (Gibco, America) supplemented with 10% inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, America), 1% nonessential amino acids (NEAA), 1% HEPES, and 500 μg/mL G418 (Gibco, America). HepG2 cells and Huh7 cells were cultured in DMEM medium, supplemented with 10% inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, America), 1% NEAA, and 1% HEPES. The transfection of siRNAs and plasmids was performed with Lipofectamine 2000 transfection reagent (Invitrogen, America) according to the manufacturer’s instructions. Peripheral blood mononuclear cells (PBMCs) from CHB patients (obtained with signed informed consent) were separated by Ficoll density gradient centrifugation and cultured in RPMI-1640 + L-glutamine medium (Gibco, America) supplemented with 10% inactivated FBS. DOTAP liposomal transfection reagent (Sigma-Aldrich) was used in the transfection of PBMCs with siRNAs.

Lan T., Wei Z., He Y., Wan S., Liu L., Cheng B., Li R., Chen H., Liu G, & Meng Z. (2021). Immunostimulatory siRNA with a uridine bulge leads to potent inhibition of HBV and activation of innate immunity. Virology Journal, 18, 37.

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Generating Bone Marrow-Derived Macrophages

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Bone marrow (BM) cells from WT mice and various mouse strains were harvested from the femurs and tibias of mice. Cells were cultured in DMEM containing 10% FBS and 30% conditioned medium from L929 cells expressing M-CSF. After 1 week of culture, non-adherent cells were removed, and adherent cells were 80%–90% F4/80⁺CD11b⁺as determined by flow cytometric analysis. LPS-primed BMDMs were infected with EHEC (MOI = 25:1), or P. aeruginosa (MOI = 30:1) for 1.5 h and then cultured in 100 μg/mL gentamycin. LPS-primed BMDMs were infected with Burkholderia thailandensis (MOI = 50:1) for 1 h and then cultured in 300 μg/mL kanamycin for another 3 h. Other stimulations included ATP (2.5 mM, 30 min), CTB (20 μg/mL, 16 h), nigericin (20 μM, 3 h). Poly (dA:dT) (1 μg/10⁶ cells, 6 h) and LPS (2.5ug/10⁶ cells) were transfected using Lipofectamine (Invitrogen). Flagellin (2 μg/10⁶ cells, 4 h) were transfected using DOTAP liposomal transfection reagent (144189731, Sigma-Aldrich). In some experiments, 1 μg of LPS was directly delivered into the cytosol of HEK293T cells or LPS-primed BMDMs by electroporation to activate Casp-11 non-canonical inflammasome, using P2 Primary Cell 4D-Nucleofector X Kit (Lonza) according to manufacturer’s instruction. Two hours post LPS electroporation, the cells were collected and lysed for Western-blot studies.

Little P., Griffin S., Kelly J., Dickson N, & Sadler C. (1998). Effect of educational leaflets and questions on knowledge of contraception in women taking the combined contraceptive pill: randomised controlled trial. BMJ : British Medical Journal, 316(7149), 1948-1952.

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