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. Hydroxychloroquine 200 mg tab Dmard therapy hydroxychloroquine Lentivirus production and transduction. Scrambled shRNA #RHS6848 and specific Nurr1 shRNA cloned into pLKO.1 lentiviral vector were purchased from GE Dharmacon. Knockdown of Nurr1 expression levels was verified by quantitative real-time PCR. 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. Leave Chloroquine on the cells for more than 24 hours due to cytotoxicity. 4Pipette up and down and carefully add don’t add too quickly or you’ll wash the cells off the plate! the 1mL of precipitated DNA to cells and incubate overnight. in 3% CO. 2. incubator 10-12 up to 24 hours from adding chloroquine. 24 Hours has been done and is fine. 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 lentivirus production Improve Lentiviral Production Using Lipofectamine 3000., Protocol for Lentivial Vector LV Production 2nd Generation. Plaquenil como funcionaLysosomotropic agent chloroquine How do you do transfection for lentivirus production. For CaPO3, I use chloroquine, which inhibits DNA degradation by lysosomes by raising their pH - but this is toxic to the cell, so media. How do you do transfection for lentivirus production?. Lentivirus Protocol. What is the best applicable inhibitor of autophagy?. If the lentivirus isn’t going to encode a fluorescent protein, cells are fixed and immunofluorochemistry is used to distinguish transduced cells. Results and Discussion. The primary measures in optimizing production of lentivirus involved rectifying steps of possible variation and excretion of components that can cause downstream toxicity. 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. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors Robert H Kutner1, Xian-Yang Zhang1 & Jakob Reiser1,2 1Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA. 2US Food and Drug Administration, Center for Biologics Evaluation and Research, Division of Cellular and.