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. Plaquenil helps hair loss Chloroquine method of action lysosome Hydroxychloroquine sulfate and hydroxychloroquine sulfate plaqueni 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. Transfection of 293T cells • Calcium Phosphate transfection reagents self-made or kits, e.g. Promega ProFection or Sigma #CAPHOS. • 25mM Chloroquine in PBS. CHLOROQUINE PHOSPHATE, USP DESCRIPTION ARALEN, chloroquine phosphate, USP, is a 4-aminoquinoline compound for oral administration. It is a white, odorless, bitter tasting, crystalline substance, freely soluble in water. ARALEN is an antimalarial and amebicidal drug. 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 High efficiency polyoma DNA transfection of chloroquine treated cells., Transfection of 293T cells - Kristoffer Riecken Hydroxychloroquine autophagy How to use Chloroquine Phosphate. Take this medication by mouth, usually with food to prevent stomach upset, exactly as directed by your doctor. Daily or weekly dosing, dosage amount, and length. Chloroquine Phosphate - WebMD. CHLOROQUINE PHOSPHATE, USP. ProFection Mammalian Transfection System - Promega. 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. This protocol is a modified version of a published method4, in which calcium phosphate–based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. Chloroquine is an inhibitor of the lysosomal degradation of the DNA which is taken up by the cells, so as leelee said, transfection should have been successful, albeit at a slightly lower level than if you would have added the chloroquine. In my normal transfections, I never add chloroquine and get high efficiencies nevertheless.