Chloroquine transfection efficiency


FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to 56 ℃ for 30 minutes. 14 Afterward, several researchers reported neomycin, 15 dexamethasone, 16 and nocodazole 17 also made a contribution to the transfection efficiency enhancement. Chloroquine Diphosphate Applications Used to study the role of endosomal acidification in cellular processes, such as the signaling of intracellular TLRs Used as DNA intercalator & to dissociate antigen antibody complexes without denaturing red blood cell antigens. Cationic lipids are frequently used as nonviral vectors to deliver DNA to cells in vitro because of their ease of use, and one of the most efficient lipids is DOTAP ]), with the result that the increased transfection efficiency seen in some cells after chloroquine treatment cannot be attributed solely to its effect on DNase II activity. Because acidic pH of endosomes is a prerequisite of endosomal TLR activation, chloroquine can serve as an antagonist for endosomal TLRs efficient methodsofgenetransferin chosentargetcells. For in vitro research. The CalPhos Mammalian Transfection Kit is a complete calcium phosphate-based kit for the transfection of mammalian tissue culture cells. Add the transfection mix dropwise being careful not to dislodge the cells. As a control, the same cell lines were transfected with polylysine in the …. (30%-70% confluent if using Optifect™ Transfection Reagent) Low transfection efficiency. P-NH2-Q was the most efficient carrier. Do not use cationic lipid reagents that have been frozen. The classical procedure makes use of retroviruses with which efficacious transfer and expression ofgenes is usually ob-tained. Using a 5 min reaction time gave intermediate results. Search. We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors the efficient miRNA delivery as higher content of chloroquine caused increased endosomal escape [ 22]. Cells are subjected to hostile conditions such as nutrient depletion and chemical or chloroquine transfection efficiency environmental stress to induce lysosomal-mediated bulk clearance of cellular debris that autophagic. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Nov 15, 2004 · Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N‐quaternized PEIs and polylysine by 2–3‐fold. [H Luthman, G Magnusson] PMID 6298741 . In particular, we have used the transferrin/. Chloroquine is an aminoquinoline that is an inhibitor of autophagy and has antimalarial and anticancer activities. Chloroquine is widely used to enhance polyplex-mediated gene transfer in vitro by favouring the survival of polyplexes in endocytic vesicles and promoting their release into the cytoplasm ( 10, chloroquine transfection efficiency 40) Transfection efficiency was analyzed by flow cytometry analysis, using FACS. Dec 14, 2015 · The ability of the polymer/pDNA complexes to escape the endosome was tested through the addition of chloroquine, a chemical known to swell and burst endosomes releasing its contents into the cytosol. However, it is worth noting that chloroquine does not always lead to an improvement of transfection efficiency, depending on the uptake pathway. In addition, chloroquine could increase the intralysosomal pH and, therefore, inhibit acid nucleases. In the presence of chloroquine, as reported earlier (5), these cells canbetransferrinfected at very high level with polyly-sine-transferrin-reporter-DNA complexes, even in the ab-. Almost 2 µg/ml for HEK-293 cells and 0.5 µg/ml for CHO cells of TNK-tPA were detected by ELISA in the …. This highly efficient vector may be of great value for the future development of gene transfer systems Scholars@Duke Search form. Used to increase transfection efficiency.For 15cm plates, add 1.69ml of the transfection buffer to the DNA-CaCl2 solution by “bubbling”. It is to be noted that the effect of chloroquine on transfection depends on the kind of cell and on the carrier Changing medium ~1 hr before transfection and supplementing the medium with 25 uM Chloroquine enhances transfection efficiency quite well. 0.01% to approx. Search. 2) Add the buffer gently to the vector mixture with shaking the tube The microtubule-depolymerizing agents colchicine, vinblastine (VB), vincristine, nocodazole, and podophyllotoxin were chloroquine transfection efficiency found to increase dramatically the transfection of cationic phospholipid–DNA (CMV–β-gal) complexes on cultured vascular smooth muscle cells (VSMCs). Volume should be 10 ml per dish. Scholars@Duke Search form. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods Transfection in the presence of N 4 - (7-trifluoromethyl-4- quinolinyl)-N 1 ,N 1 -diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at-same concentration, while transfection in the presence of N 4 - (4- quinolinyl)-N 1 ,N 1 -diethyl-1,4-pentanediamine. chloroquine transfection efficiency …. Weand others (1, 2) have explored the alternative methodoftransferring genes byreceptor-mediated endocy-tosis (3-5). This reduction in transduction efficiency remained in the generations of chloroquine transfection efficiency chloroquine transfection efficiency cells, even after the disappearance of conditions causing cellular toxicity Chloroquine has been used to increase transfection efficiency for calcium phosphate, DEAE-dextran, and some liposome-mediated gene delivery [63,65–68]. Addition of chloroquine ("lysosomotropic" agent) to transfection medium substantially enhanced the HEK293 transfection. In contrast, chloroquine did not improve the transfection efficiency of PEI. Luthman H, Magnusson G. The following morning, carefully aspirate the media. Invitrogen product line such as Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent To date, chloroquine has been widely used to elucidate the uptake mechanism of non-viral nucleic acid delivery systems (Legendre and Szoka Jr 1992; Simeoni, Morris et al. Other evidence comes from experiments in which reputed inhibitors of DNase II increased transfection efficiency of cells. In cell culture lab, it helps to improve transfection efficiency and gets more and more into the focus of cancer treatment. Add 1ml of 2x sterileHBS drop-wise to the mixture, while gently vortexing the tube. The kit contains reagents necessary to perform 100 transfections in 10 cm plates or 700 transfections (6-well plate format) US9102796B2 US12/333,453 US33345308A US9102796B2 US 9102796 B2 US9102796 B2 US 9102796B2 US 33345308 A US33345308 A US 33345308A US 9102796 B2 US9102796 B2 US 9102796B2 Authority US United States Prior art keywords cell composition nucleic acid polymer php Prior art date 2007-12-12 Legal status (The legal status is an assumption and is not a legal …. DNA delivered by Ca2PO4 transfection is thought to …. Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. 293T cells 2),which were made by introducing SV40 T antigen gene into 293 cells, are useful for transient transfection …. Improper cell density. 7). The development of reporter gene systems and selection methods for stable maintenance. Replace the media with 15 mL of DMEM complete Actually there is no rule at all for transfection efficiency: depending on the cell type (clone), plasmid design, plasmid purification (presence of salts), level of coil, strength of the promoter, medium, kinetics of experiments, read out, () efficiency can vary from one lab to another In a study by Lung et al., sequences of histidine and cysteine residues incorporated into the TAT structure which led to promote the gene transfection efficiency by up to 7000-fold over the original Tat peptide Since the publication of the original method 1, increases in efficiency have been achieved by including additional steps such as glycerol shock 2 and/or chloroquine treatment 3 Transfection efficiency was determined by staining β-galactosidase expressing cells with X-Gal after 24 h . Transfection efficiency was analyzed by flow cytometry analysis, using FACS. Figure 6.1 Cell line-dependent differences in transfection efficiency. The insoluble precipitate attaches to the cell surface and is brought into the cells by endocytosis. The inability of polyplexes to avoid this fate is chloroquine transfection efficiency a major barrier to efficient transfection following internalization by endocytosis ( 10). 2010). 2 a–c). 2003; Lehto, Abes et al. In cell culture lab, it helps to improve transfection efficiency and gets more and more into the focus of cancer treatment Aug 31, 2017 · Furthermore, the transfection reagent might be critical in chloroquine’s effect. 2010). Abstract Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. It is to be noted that the effect of chloroquine on transfection depends on the kind of cell and on the carrier Figure 6.1 Cell line-dependent differences in transfection efficiency. Invitrogen product line such as Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent Transfection in the presence of N 4 -(7-trifluoromethyl-4-quinolinyl)- N 1, N 1 -diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N 4 -(4-quinolinyl)- N 1, N 1 -diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to. Chloroquine are known to help the DNA escape into the cytoplasm by disrupting endosomes and sabotaging the endosomal transport pathway. histones were used to complex the DNA, transfection efficiency increased over 10-fold to a …. The topology (linear or supercoiled) and the size of the plasmid chloroquine transfection efficiency DNA vector influence the efficiency of transfection. In contrast, chloroquine did not improve the transfection efficiency of PEI. falciparum (IC 50 = 29.2 nM) but has decreased activity against mutant pfcrt P. High efficiency polyoma DNA transfection of chloroquine treated cells. In some cases, cells were treated with 100 µM chloroquine when transfected with PEI 2 …. (30%-70% confluent if using Optifect™ Transfection Reagent) Low transfection efficiency Chloroquine is used for treating amebiasis, rheumatoid arthritis, discoid and systemic lupus erythematosus. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency If chloroquine is not used, it is unnecessary to change the medium prior to transfection. 1,2,3 Chloroquine inhibits autophagosome-lysosome fusion in HeLa cells when used at a concentration of 100 µM. of fluorescence intensity. Cationic lipid was frozen.