The soluble complex is added to the cells, which take it up by nonspecific endocytosis. The method described here uses an initial incubation with a DNA/DEAE-dextran mixture, followed by a second incubation in growth medium supplemented with chloroquine. Is there withdrawal from hydroxychloroquine Chloroquine and filipin lysosome Transfection Mediated by DEAE-Dextran High-Efficiency Method Protocol summary only for purposes of this preview site Transfection mediated by DEAE-dextran differs from calcium phosphate coprecipitation in three important respects. First, it is used for transient expression of cloned genes and not for stable transformation of cells. A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A coincubation of the cells with chloroquine during. Likewise, chemical methods used to introduce DNA using DEAE-dextran can become difficult because endocytotic mechanisms that uptake these reagents can accumulate in toxic quantities in the cell. The result is often varying transfection efficiencies which have been found to produce as low as 10% or less successful gene transfer in primary cells. This protocol describes transfection of lymphocyte cell lines, but it can be used for transient transfection of both adherent and nonadherent cells. Chloroquine is thought to enhance transfection efficiencies by neutralizing lysosomal hydrolases, which can degrade the DNA. Deae dextran chloroquine method Transfection Methods Overview - Bio-Rad, DEAE-Dextran transfection Request PDF Otis pregnancy plaquenilHydroxychloroquin eye Methods are described for achieving high efficiency transient transfection of COS-1 cells. A transfection solution of vector DNA and DEAE-dextran in phosphate buffered saline is added to the cells, followed by treatment with culture medium containing chloroquine, resulting in a maximum efficiency of about 50%. High efficiency transfection of monkey kidney COS-1 cells.. Monitoring Autophagy in Live Cells Without Transfection.. Gene transfer by electroporation, lipofection, and DEAEâ.. Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. The DEAEdextran-chloroquine shock method17, 18 with 25,gofcesiumchloride gradient-purified DNAand 1.5 mg of DEAE dextran per 10-cm Petri dish. Cells were then incubatedfor 3 hrat 37°C in 6mlofDMEMcontaining 300,tgofchloroquine, washed,andcultured in DMEMfor48or tTowhomreprint requests should beaddressed. To examine the localization of mutant sortilin, we transiently transfected COS-7 cells with the pcDNA3.1 vector using a DEAE/Dextran chloroquine phosphate technique. Cells were then incubated for 48 hours to allow the mutant protein to be expressed.