Chloroquine transfection protocol

Discussion in 'Chloroquine Phosphate' started by Se0man, 17-Mar-2020.

  1. scriptone Guest

    Chloroquine transfection protocol


    This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation.

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    Prior to adding complex, remove 1 mL of medium from each well, leaving a total of 1 mL in each well. Add 500 μL of lipid–DNA complex to each well, taking care to dispense liquid against the well wall to avoid disrupting cells. Gently agitate plate to evenly distribute. Incubate plate for 6 hours at 37°C, 5% CO 2. Day 2 Phoenix cell transfection & target cell preparation Part 1 Phoenix cell transfection 1. Feed Phoenix cells with DMEM supplemented with chloroquine diluted from 1000x stock to 1x 30 min before transfection. 2. In a FACS tube in the hood, add the following 15 µg DNA 40 µL 2M CaCl 2 MilliQ water to final volume of 500 µL 3. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. 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.

    Test a variety of brands and lots of FBS to find one suitable with your protocols. Last Upload: August 26, 2016 *Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection.

    Chloroquine transfection protocol

    Transient Mammalian Cell Transfection with., Retrovirus preparation using Phoenix cells

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  4. This protocol is a modified version of a published method 4, in which calcium phosphate–based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells.

    • Calcium phosphate–mediated transfection of eukaryotic..
    • Addgene General Transfection.
    • Evaluation of Methods for Transient Transfection of a..

    Transfection Experiment Transfection Efficacy Reagent Volume Tissue Culture Grade Chloroquine Diphosphate These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves. Plate cells the night before to give 60-80% confluence at the day of transfection. 2. 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. Chloroquine is used for treating amebiasis, rheumatoid arthritis, discoid and systemic lupus erythematosus. Application DNA intercalator. Also used to increase transfection efficiency. Chloroquine diphosphate salt has been used • in in vitro antiplasmodial assays • in transfection and infection assays • in autophagy inhibition

     
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