Chloroquine in transfection

Discussion in 'Chloroquin' started by gruppastimul, 01-Mar-2020.

  1. favourusiess Well-Known Member

    Chloroquine in transfection


    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.

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    May 07, 2001 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. Chloroquine-resistant cells efflux chloroquine at 40 times the rate of chloroquine-sensitive cells; the related mutations trace back to transmembrane proteins of the digestive vacuole, including sets of critical mutations in the P. falciparum chloroquine resistance transporter PfCRT gene. 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 in transfection

    Evaluation of Methods for Transient Transfection of a., Chloroquine - Wikipedia

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  4. PKa 8.4 and 10.8 for chloroquine base at 20 °C 2. This product is a member of the quinoline family with multiple applications. It was originally used as an antimalarial compound.3 It kills the erythrocytic forms of malaria parasites at all stages of development, but does not affect the sporozoites.

    • Chloroquine diphosphate salt - Sigma-Aldrich.
    • Chloroquine Diphosphate 100 mm for Transfection.
    • Transient Transfection of 293T cells.

    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. Transfection Methods Reagent-Based Methods DEAE-Dextran Method Overview Solution A DNA ~1–5 µg/ml diluted into 2 ml of growth medium with serum containing chloroquine Solution B DEAE-dextran solution ~50–500 µg/ml Solution C ~5 ml of DMSO Solution D Complete growth medium 1 Add solution A to solution B, then mix gently. One hour prior to the transfection, change to medium containing 25µM chloroquine from x1000 stock in PBS, stored at -20°C. Volume should be 10 ml per dish. Chloroquine can be omitted, but increases efficiency about x2 3. Add 10µg DNA and ddH2O to 438 µl in 15-ml sterile tube.

     
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