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 for sun allergy Coppolino quarantine chloroquine Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15-20 min at room temperature. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells. Add DNA/CaCl2-solution to 2x HBS drop wise, while blowing air through HBS with pasteur pipette. • Incubate the mixture for 10 to 20min at room temperature. • Remove old medium from the cells. • Add 10ml medium including 25µM Chloroquine the Chloroquine is used in this step only. 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. 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 Glycerol shock or chloroquine? - Transfection and Transduction, Transfection of 293T cells - Kristoffer Riecken Plaquenil 400 mg Typically, DNA is suspended in sterile water or TE buffer to a final concentration of 0.2–1 mg/ml The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA, transfection reagent/method, target cell line, and number of cells Genetic Material DNA Quality and Quantity Factors Affecting Transfection Methods Overview - Bio-Rad. Structure−Function Correlation of Chloroquine and Analogues.. Transient transfection into 293T cells by Jun Takagi, Jun 15.. Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. 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-mediated toxicity usually leads to changes in gene expression, even in the absence of any visual toxicity. Therefore, it is important to choose a transfection method that can mitigate both observed as well as non-apparent cellular toxicity. Cationic lipids and polymers also form complexes with DNA through electrostatic interactions.