Chloroquine in transfection

Discussion in 'Discount Prescriptions' started by Saniok, 05-Mar-2020.

  1. mads XenForo Moderator

    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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    Calcium phosphate co-precipitation as a transfection method was introduced in the early 1970s Graham and van der Eb, 1973. The method is still popular because the components are easily available and inexpensive, the protocol is easy to use, and it is effective with many different types of cultured cells. Addition of chloroquine improves transfection efficiency and subsequent virus titer, about 3- to 5-fold, by stabilizing cell lysozomes and increasing the fraction of DNA that reaches the nucleus. Sodium butyrate another lysosome stabilizer has also been used for this purpose. 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.

    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

    Calcium phosphate–mediated transfection of eukaryotic cells., Production of Replication-Defective Retrovirus by Transient.

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  7. 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.

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    So we use Chloroquine as a transfection agent at the concentration 25uM for 8 hours. Than changing the media. So far we detected much higher transfection efficiency with Chloroquine than. 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. 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.

     
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