This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Rheumatoid arthritis guidelines hydroxychloroquine monotherapy Plaquenil derived from Plaquenil skin itching Medicine for lupus plaquenil Prepare the DNA in HBS for application to cells. 1. About 5 minutes prior to transfection, add chloroquine to each plate to 25uM chloroquine stock is 50 mM; for 3 mL media + 1 mL DNA, add 2u1. Chloroquine acts to inhibit lysosomal DNases by neutralizing vesicle pH. Protocol for Lentivial Vector LV Production 2nd Generation Packaging Lenti virus can be packaged by any transfection reagent. In this protocol, we describe two methods a calcium phosphate precipitation method and an iMFectin Poly DNA method. When performing a transient transfection, you can choose between a standard or reverse transfection protocol. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and. Test a variety of brands and lots of FBS to find one suitable with your protocols. Last Upload: August 26, 2016 *Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection. Chloroquine transfection protocol Chloroquine diphosphate salt powder or crystals, 98.5-101.0%., Protocol for Lentivial Vector LV Production 2nd Generation. Chloroquine mechanismHydroxychloroquine sle mechanismPlaquenil and high blood sugarIs plaquenil covered by blue crossChloroquine mechanism of action slideshare I have different protocols for Lentivirus production using Lipofectamine transfection of HEK293T cells and now am confused about the concentration of Chloroquine and Na-Butyrate. Questions with answers in CHLOROQUINE Science topic. Transfection Guide Overview of Transfection Methods Promega. Addgene General Transfection. Chloroquine diphosphate 52 mg Deionized, distilled H 2 O 1 mL Dissolve 52 mg of chloroquine diphosphate in 1 mL of deionized, distilled H 2 O. Sterilize the solution by passing it through a 0.22-µm filter. Store the filtrate in foil-wrapped tubes at −20°C. The following are the protocols I've gathered *4-6 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. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80°C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.