If you use 6-well or 12-well plates, total volume of the medium should be 3ml and 1.5ml, respectively, and decrease the amount of each reagent accordingly.2. One hour prior to the transfection, change to medium containing 25M chloroquine (from x1000 stock in PBS, stored at -20C). (Chloroquine can be omitted, but increases efficiency about x2) to a final volume of 500 l. Lysosomal inhibitor chloroquine 2016 ophthalmology journal hydroxychloroquine Discoid lupus plaquenil Chloroquine diphosphate CQ was ingeniously used to take place of phosphate salt in traditional calcium phosphate coprecipitation method for pDNA transfection. With multiple roles of CQ in the novel Ca-CQ-pDNA complex including pDNA compaction and assistance in lysosome escape, the transfection efficiency of the pDNA was significantly increased relative to the traditional method. CQ did not. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine 100 microM for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene. Transfection of 293T cells • Calcium Phosphate transfection reagents self-made or kits, e.g. Promega ProFection or Sigma #CAPHOS. • 25mM Chloroquine in PBS. Add this mixture directly to the cells dropwise through the medium. As long as the cells are alive, they produce proteins. When you are ready, add 500 l of 2x HBS dropwise while gently mixing. Make sure you evenly sprinkle the droplet over the entire area.5. For secreted protein, you can change media once (at day 3 or 4, save the collected sup) and give it another 3-4 days of culture. Chloroquine transfection calcium phosphate Transfection - Wikipedia, High-efficiency stable gene transfection using chloroquine-treated. Is plaquenil safer than methotrexateInteraction between hydroxychloroquine and methotrexateHydroxychloroquine and glaucomaHydroxychloroquine increase metroxtratePlaquenil use for lupus 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. High efficiency polyoma DNA transfection of chloroquine treated cells.. Transfection of 293T cells - Kristoffer Riecken. Plasmid transfection in HEK293T cell by Calsium Phosphate.. I have another question regarding calcium phosphate transfections. I am planning to do another this week, but I would like to compare standard CaPO4 transfection efficiencies w/those of modified techniques. I've read several articles suggesting a glycerol shock and/or adding chloroquine to the media. Phosphate solution, and allowing the mixture to incubate at room temperature. This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. Reagent-Based Methods Calcium Phosphate Ca++ Ca++ Ca++ Ca++ Calcium phosphate Ca++ Ca++ DNA Common Transfection. Add 22 l of 25mM chloroquine to each 15cm dish for 10cm plate 7.8 l of chloroquinei.e. final concentration = 25 M. The cells should be transfected within 5-10’ of adding the chloroquine. 7. For 15cm plates, add 1.69ml of the transfection buffer to the DNA-CaCl2 solution by “bubbling”.