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. What are some side effects of hydroxychloroquine Plaquenil plm Calcium phosphate transfection is one of the most commonly used methods to deliver plasmid DNA into mammalian cells. Due to its highly consistent pH and salt concentration, our CalPhos calcium phosphate transfection kit gives you the consistent transfection efficiency that you cannot always achieve with home brew versions. Chloroquine enters the red blood cell, inhibiting the parasite cell and digestive vacuole by simple diffusion. Chloroquine then becomes protonated to CQ2+, as the digestive vacuole is known to be acidic pH 4.7; chloroquine then cannot leave by diffusion. 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. 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 Methods Overview - Bio-Rad, Chloroquine - Wikipedia Hydroxychloroquine hypertension Jul 30, 2019 Before the administration of chloroquine, the patient had only a mild skin erythema in the irradiated area, which was consistent with the radiotherapy dose she had received. On day 3 of chloroquine therapy, she developed localized brisk bullous eruptions in the irradiated area, which developed into a patch of fulminant moist desquamation. Chloroquine Side Effects Common, Severe, Long Term -. High-efficiency stable gene transfection using chloroquine-treated.. Plasmid transfection in HEK293T cell by Calsium Phosphate.. Transfection can be carried out using calcium phosphate i.e. tricalcium phosphate, by electroporation, by cell squeezing or by mixing a cationic lipid with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. Transfection can result in unexpected morphologies and abnormalities in target cells. Calcium phosphate transfection is the method of choice to produce long-term stable transfectants. This method also works well for transient expression of transfected genes and can be used with most adherent cell lines. 2. Product Components and Storage Conditions Product Cat.# ProFection® Mammalian Transfection System—Calcium Phosphate E1200 The Calcium Phosphate Transfection Kit provides high-quality reagents to enable the introduction of DNA into eukaryotic cells via calcium phosphate co-precipitation. How it works The calcium phosphate transfection method for introducing DNA into mammalian cells is based on forming a calcium phosphate-DNA precipitate.