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. Does prednisone and plaquenil work for ankylosing spondylitis Hydroxychloroquine 200 mg dosage Chloroquine use and fluoroquinolone resistance dissertation 1h chloroquine binding biochemistry and biophysics Hour before transfection, replace cDMEM with cDMEM + 25 mM chloroquine. Add transfection reaction to this media, and incubate o/n JoVE. *After "DNA absorption" 4 h again. replace transfection reaction-containing media with cDMEM + 100 mM chloroquine. Change media after 2-5 hours Luthman & Magnusson, 1983. After cells chloroquine-treated or untreated have been exposed to the calcium phosphate–DNA coprecipitate in growth medium for 2–6 h, remove the medium by aspiration and wash the. 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. 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 dna transfection High efficiency polyoma DNA transfection of chloroquine., Calcium phosphate–mediated transfection of eukaryotic cells. Plaquenil hair growthChloroquine phosphate brooklynella Quinacrine QC, a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N4-4-pyridinyl-N1,N1-diethyl-1,4-pentanediamine CP, a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Structure−Function Correlation of Chloroquine and Analogues.. Chloroquine Diphosphate 100 mm for Transfection. Putative Role of Chloroquine in Gene Transfer into a Human.. The total volume of transfection complex medium, DNA and ViaFect™ Transfection Reagent to add per well of a 96-well plate is 5–10μl. The following protocol is a guideline for transfecting approximately 10–20 wells, depending on the volume of ViaFect™ Transfection ReagentDNA mixture used. For additional wells, scale volumes accordingly. Transfection Procedure. The morning of the transfection day, replace the media with fresh DMEM without PSG and containing 10 uL of 25 mM chloroquine optional. Wait ~5h before going onto the next step. Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well. Add 3 uL PEI per ug of DNA in OptiMEM. A pH-responsive peptide fragment modelled on the influenza virus haemagglutinin INF7-SGSC can promote the transfectional activity of polyL-lysine pLL/DNA complexes against 293 cells.