The Lipofectamine RNAiMAX reagent protocol (Thermo Fisher) was used to transfect cells with siRNA. Briefly, 0.75 × 106 THP1 cells (50%-confluent) were transfected with the complexes containing 10 nM siRNA and 6 μl Lipofectamine RNAiMAX in 150 μl of Opti-MEM medium. The cells were incubated with siRNA–lipid complexes for 4 h, after which the mixture was aspirated and replaced with 1 ml of complete culture medium. Poly(I:C) (1 μg) was transfected into the cells with 6 μl Lipofectamine 3000 (Thermo Fisher) in 150 μl of Opti-MEM medium, according to the manufacturer protocol. Subsequent steps were the same as described above. For the transfections with the DOTAP Liposomal Transfection Reagent (Roche), 1 μg poly(I:C) was mixed with 8 μl of HBS buffer (20 mM HEPES, cell culture grade, 150 mM NaCl, pH 7.4) and 6 μl of DOTAP in 12 μl of HBS buffer. The cells were mixed with transfection complex in 0.3 ml of Opti-MEM medium, incubated for 4 h at 37°C and then cultured in the regular media for 48 h.
Dotap liposomal transfection reagent
Manufactured by Roche
Sourced in France
DOTAP Liposomal Transfection Reagent is a cationic lipid-based reagent used for the delivery of genetic material, such as DNA or RNA, into cells. It is designed to facilitate the efficient transfection of a variety of cell types, enabling the study of gene expression and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by InvivoGen (3 mentions)
Selection for hygromycin resistance, by Thermo Fisher Scientific (2 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Transit 2020 reagent, by Mirus Bio (2 mentions)
Dotap reagent, by Roche (2 mentions)
Lipofectamin 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Recombinant dnase 1, by Roche (2 mentions)
Chloroquine, by InvivoGen (2 mentions)
Cu cpt9a, by InvivoGen (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax reagent protocol, by Thermo Fisher Scientific (1 mentions)
5 ppp dsrna, by InvivoGen (1 mentions)
On targetplus smartpool, by Horizon Discovery (1 mentions)
Dotap, by Roche (1 mentions)
Pcohygro, by Thermo Fisher Scientific (1 mentions)
Deltavision microscope, by Cytiva (1 mentions)
Cpg a 2216, by InvivoGen (1 mentions)
Gw4869, by Merck Group (1 mentions)
Psiren retroq vector, by Takara Bio (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
31 protocols using dotap liposomal transfection reagent
1
Transfecting THP1 Cells with siRNA and poly(I:C)
2
Peptide and RNA Vaccine Formulation
3
Silencing Host Factors Regulates ZIKV Infection
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Selected siRNA molecules (On-TARGET plus SMARTpool, Dharmacon) were transfected using the DOTAP liposomal transfection reagent (Roche, Switzerland), as described in a previous study67 (link). Briefly, human pDCs were isolated from healthy donors by negative selection using the plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec, Germany), with a purity of >95%, as determined by flow cytometry (Supplementary Fig. 1c ). Isolated pDCs were seeded at 105 cells/100 μL in 96-well plates and incubated at 37 °C. siRNAs were diluted in PBS for a final concentration of 160 nM and mixed with 1:1 with DOTAP (Roche). The mix was incubated at room temperature for 15 minutes and then added to cells and incubated for 24 h without medium change. After 24 h post transfection, a small cell sample was collected to check the gene silencing efficacy by qPCR; the remaining cells were infected with ZIKV at a MOI of 1. At defined time points after infection, total ZIKV RNA was quantified by quantitative RT-PCR (qRT-PCR) as described in our previous study16 (link).
4
Transfection, RNAi, and Irradiation Protocols
5
HMGB1 Gene Silencing in Bladder Cancer Cells
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The sequence of small interfering RNA (siRNA) targeting HMGB1 was designed by an RNA interference activity-predicting algorithm and synthesized by Eurogentec (Seraing, Belgium). The sequences HMGB1 siRNAs are as follows: 5′-GATCCGCTGAAAAGAGCAAGAAAATTTCAAGAGAATTTTCTTGCTCTTTTCAGCTTTTTGGAAG-3′ (sense); 5′-AGCTCTTCCAAAAAGCTGAAAAGAGCAAGAAAATTCTCTTGAAATTTTCTTGCTCTTTTCAGCG-3′ (antisense). One scrambled siRNA (Scramble II; MWG Biotech) was used as a control. After seeding and adherence for 24 h, BIU-87 and T24 cells were transfected in serum-free OptiMEM with 200 nM of HMGB1 siRNA using DOTAP liposomal transfection reagent according to the manufacturer’s instructions (Roche, Mannheim, Germany). Following transfection (4 h, 37°C), cells were washed with phosphate-buffered saline (PBS) and incubated in serum-containing medium for 72 h. For further analyses, cells were harvested by trypsin treatment (0.05% trypsin/0.02% EDTA, 5 min, 37°C). Detached and adherent cells were pooled and analyzed together. The expression levels of HMGB1 in siRNA-treated cells were examined by Western blotting.
6
Transfection of BMDMs with LPS and LPGs
7
Synthetic TLR9 Ligand CpGA 2216 Protocol
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Mice were injected i.v. with 10 μg/mouse of the synthetic TLR9 ligand CpGA 2216 (Invivogen, France), complexed with DOTAP liposomal transfection reagent (Roche Applied Sciences, France), and sacrificed 7 h after injection. To block IFNAR1, mice were treated 24 h before sacrifice with 1 mg of isotype control or anti‐IFNAR1 MAR‐1 mAbs (BioXcell, USA).
8
TLR8 Overexpression and miRNA Modulation
9
Extracellular Vesicle Isolation and Characterization
10
Generation of Retroviral Vectors for Fas Knockdown and FasL Expression
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A cDNA encoding a membrane-bound form of HEL (mHEL) excised from pcDNA3-mHEL (a gift of Dr. R. Brink [19] (link)) was inserted into pMX-IRES-GFP [34] (link) to make pMX-mHEL-IRES-GFP. An shRNA sequence targeting the Fas 3′UT sequence, 5′-gtgttctctttgccagcaaat-3′, was inserted into pSIREN-RetroQ vector (Clontech), to make a retroviral vector pSIREN-RetroQ-shFas. An eGFP sequence in the pMX-IRES-GFP vector was replaced with a cDNA consisting of extracellular and transmembrane domains of human CD8 (hCD8) to make pMX-IRES-hCD8. A FasL cDNA was inserted into the pMX-IRES-hCD8 to make pMX-FasL-IRES-hCD8. The retroviral vectors were transfected into packaging cells, PLAT-E [34] (link), using FuGENE (Roche). On the next day, the supernatants were added to target cells in the presence of 10 μg/mL DOTAP Liposomal Transfection Reagent (Roche).
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