HEK293T cells (ATCC, CRL-11268) were maintained at 37 °C in 5% CO2 atmosphere in DMEM High Glucose with 4.5 mM L-glutamine (GE Life Sciences, SH30003.03) supplemented with 1 mM sodium pyruvate (Alfa, A11148), 10% fetal bovine serum (Sigma, F0926), and 1% penicillin-streptomycin (Corning, 30-002-CI). For a 96-well format used in all imaging-based experiments, 100 µL of media was used for culturing, whereas for a 12-well plate used for western blot or biochemical assays, 1 mL of media was used. Cells were monitored every 3 months to confirm the absence of mycoplasma contamination (Genlantis, MY01100). Cells were transiently transfected with LPEI (Polysciences, 23966) using a 0.5 mg/mL solution at a 5:1 LPEI:DNA (w/w) ratio in antibiotic-free DMEM. Following overnight transfection, the media was removed and the cells were gently washed once with regular DMEM to remove any excess unnatural amino acid. Cells were serum-starved (DMEM with 0.1% FBS, without antibiotics) for 4 h. The media was replaced with 90 µL of LCIS immediately prior to irradiation. Irradiation was performed on a UV transilluminator (VWR Dual Transilluminator at 365 nm) for two minutes (unless specified otherwise).
L pei
Manufactured by Polysciences
L-PEI is a linear polyethylenimine-based polymer that can be used as a transfection reagent for introducing nucleic acids into cells. It facilitates the uptake of DNA or RNA into cells by forming complexes with the genetic material. The product is suitable for a variety of cell types and can be used for both transient and stable transfection applications.
Automatically generated - may contain errors
Lab products found in correlation
Arx 300 nmr spectrometer, by Bruker (2 mentions)
Branched polyethylenimine, by Polysciences (1 mentions)
Chitosan, by Polysciences (1 mentions)
Hmw chitosan, by Merck Group (1 mentions)
Diethylaminoethyl dextran, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Gelatin, by Merck Group (1 mentions)
Poly allylamine hydrochloride, by Merck Group (1 mentions)
Linear polyethylenimine, by Polysciences (1 mentions)
L glutamine, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Poly l ornithine hydrobromide, by Merck Group (1 mentions)
Poly l lysine hydrochloride, by Merck Group (1 mentions)
Litesizer 500, by Anton Paar (1 mentions)
8 protocols using l pei
1
Optimized HEK293T Cell Culture and UV Irradiation
2
Producing Virion-Packaged HIV-1 Stocks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sucrose-cushion-purified HIV-1 stocks were produced as previously described45 . HIV-1* GFP virus stocks, carrying virion-packaged Vpx, were produced by co-transfection of the proviral HIV-1* GFP DNA and the indicated Vpx expression constructs7 (link). In brief, 293 T cells were seeded at a density of 8 × 106 cells in a 15-cm dish. After 24 h, cells were co-transfected with a mixture of 37.5 µg HIV-1 plasmid and 112.5 µl of L-PEI (3 µl of L-PEI for every µg of DNA, stock concentration of 1 µg µl−1; Polysciences) in DMEM without any additives for 30 min. After this time, the DNA/PEI solution was added to the cells. After 72 h, the supernatant was collected and virus was purified via sucrose-cushion centrifugation. For virions to incorporate Vpx, the transfection was performed as described above, but using 37.5 µg of HIV-1* GFP or HIV-1* BFP together with 18.75 µg of pcDNA-Vpx (SIVmac239-Myc) or the corresponding pcDNA3.1 empty control vector. For virus production for the virion fusion assay, the transfection was performed as described above, combining 37.5 µg of X4 HIVivo and 12.5 µg of pCMV-BlaM-Vpr.
3
Cationic Polymer Library for Gene Delivery
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Linear-polyethylenimine 22 kDa (L-PEI) (from Stéphane Bellemin-Laponnaz Laboratory, IPCMS Strasbourg), 25 kDa branched-PEI (B-PEI) (#408727, Sigma-Aldrich, St. Louis, MO, USA), Poly-L-Lysine hydrochloride (#P9404, Sigma-Aldrich), Poly-(Lys,Trp) (#P-9285, Sigma-Aldrich), Poly-L-ornithine hydrobromide (#P3655, Sigma-Aldrich), DGL-NH2G4 (Colcom). Poly-Arginines (pArg) of 10, 30, 70 and 200 (#000-R010-102, #000-R030-104, #000-R070-101, #000-R200-102) were obtained from Alamanda Polymers. Poly-L-Arginine 120 (#P7762, Sigma-Aldrich), 0.7 kDa B-PEI (#40871-9, Sigma-Aldrich), 1.8 kDa B-PEI (#40528, Alfa AESAR), 2 kDa B-PEI (#408700, Sigma-Aldrich), 750 kDa B-PEI (#18,197-8, Sigma-Aldrich), 0.73 kDa L-PEI (obtained from Luc Lebeau laboratory, Strasbourg) and 4 kDa L-PEI (#24885-2, Polysciences). Linear polyethylenimine HCl, Mw 4000 (L-PEI HCl 4000) is a fully hydrolysed (deacylated), highly water-soluble hydrochloride salt form of the Linear polyethylenimine of MW 2500. All polymers were resuspended in sterile water and kept at −20 °C.
4
Production of Purified HIV-1 Stocks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To produce sucrose cushion-purified HIV-1 stocks.87 (link) 293T cells were seeded at a density of 8 × 106 cells in a 15-cm dish. After 24 h, cells were co-transfected with a mixture of 37.5 μg HIV-1 plasmid and 112.5 μL of L-PEI (3 μL of L-PEI for every μg of DNA; stock concentration of 1 μg/μL, Polysciences, Inc) in DMEM without any additives for 30 min. After this time, the DNA/PEI solution was added on top of the cells. After 72 h, the supernatant was harvested and virus was purified via 25% sucrose-cushion centrifugation. For virus production for the virion-fusion assay, the transfection was performed as described above, combining 37.5 μg of X4 or R5 HIVivo and 12.5 μg of pCMV-BlaM-Vpr.
5
Synthesis and Characterization of LCA-LPEI Conjugate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LCA-LPEI conjugate (3 molar e quivalent LCA to 1 mole LPEI, or 3E) was synthesized using carbodiimide chemistry. Forty micromolar LPEI (base form, Polysciences) and 120 μmol LCA were dissolved in 25 mL of dichloromethane (DCM). Dicyclohexyl carbodiimide (DCC) 120 μmol and N,N-diisopropyl-ethylamine (DIPEA) 120 μL were added to the mixture and reacted over 15 h. 3E was purified by dialysis against 95% ethanol followed by dialysis against acidified DI water. The resulting product was dissolved in D2O and analyzed with a Bruker ARX-300 NMR spectrometer equipped with a 5 mm QNP probe.
6
Polymer-based Gene Delivery Agents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Linear polyethylenimine (LPEI, Mw of 2.5K, 25K, 250K) and branched polyethylenimine (BPEI, Mw of 1.2K, 10K, 50–100K) were purchased from Polysciences; low molecular weight chitosan (LMW chitosan, Mw of 15–30K), high molecular chitosan (HMW chitosan, Mw of 300K), gelatin (pH = 2.5), diethylaminoethyl-dextran (DEAE-Dextran), protamine sulfate (PrS), poly-l -lysine (PLL, Mw of 15–30K and 30–70K), and poly(allylamine hydrochloride) (PAH, Mw of 60K) were purchased from Sigma-Aldrich, and the degradable poly(beta-aminoesters) (designated Poly 1 and Poly 2) were synthesized using the method that was previously published.56
7
Synthesis and Characterization of LCA-LPEI Conjugate
8
Optimizing DNA/PEI Particle Characteristics with HA Coatings
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!