Transfections allow for transient expression of a gene of interest in a target cell line and can be useful for short term studies of protein function. We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors. Plaquenil dosage Hydroxychloroquine induced cardiomyopathy in systemic lupus erythematosus Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to 56 ℃ for 30 minutes. 25 mM chloroquine diphosphate Dissolve 0.129 g of chloroquine diphosphate salt in 10 mL of sterile water. So we use Chloroquine as a transfection agent at the concentration 25uM for 8 hours. Than changing the media. So far we detected much higher transfection efficiency with Chloroquine than. Chloroquine has been frequently used in cell culture gene transfection studies Cotton et al. 1992, Wagner et al. 1992, Lucas et al. 1995, but has not been reported yet to be successful for enabling gene transfection in vivo. As mentioned above, the mechanisms of action of chloroquine seems to be related to elevating the intravesicular pH of lysosomes and endosomes, thereby providing a suboptimal pH environment for the various lysosomal enzymes including those involved in DNA degradation. Last Upload: June 10, 2016 Day 0: Seed Lenti-X 293T cells (this cell line is optimized for production of lentiviral vectors) Day 1 (pm): Transfect Cells Day 2 (am): 18h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment *Pro-Tips* Different brands and lots of FBS can promote or inhibit transfection. This approach can be adapted for different cell lines and different transfection reagents. Chloroquine in transfection Chloroquine Diphosphate 100 mm for Transfection, Questions with answers in CHLOROQUINE Science topic Reddit plaquenil side effects Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells. Incubate the cells for 18 h, or until the following morning. The following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete. Addgene General Transfection. Enhanced plasmid DNA transfection with lysosomotropic.. Chloroquine diphosphate salt powder or crystals, 98.5-101.0%.. Transfection is the process of introducing nucleic acids into cells. In cell-based assays, transfection is typically used to investigate protein expression and gene function by introducing nucleic acids transiently or permanently into the cell and studying how the cell’s internal machinery modulates gene expression in response. Chloroquine is an aminoquinoline that is quinoline which is substituted at position 4 by a 5-diethylaminopentan-2-ylamino group at at position 7 by chlorine. It is used for the treatment of malaria, hepatic amoebiasis, lupus erythematosus, light-sensitive skin eruptions, and rheumatoid arthritis. Hours after transfection reaction has been applied to cells, gently remove media and replace with 10% glycerol or DMSO for 2-3 minutes. Remove and feed with complete media various references. *1 hour before transfection, replace cDMEM with cDMEM + 25 mM chloroquine. Add transfection reaction to this media, and incubate o/n JoVE.