Chloroquine in lentiviral transduction

Discussion in 'Without A Doctor Prescription' started by sercas, 12-Mar-2020.

  1. wings Well-Known Member

    Chloroquine in lentiviral transduction


    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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    The first sucessful report utilizing lentiviral vectors in NK cells described the transduction of primary murine NK cells. As the lentiviral transduction system allows efficient and stable integration of transgenes, NK cell lines can be utilized to develop in vitro models to study and further understand the biology of NK cells. Add 22 l of 25mM chloroquine to each 15cm dish for 10cm plate 7.8 l of chloroquinei.e. final concentration = 25 M. The cells should be transfected within 5-10’ of adding the chloroquine. 7. For 15cm plates, add 1.69ml of the transfection buffer to the DNA-CaCl2 solution by “bubbling”. Optimization of DNA amounts and N/P ratios for lentiviral vector production in DMEM medium lacking serum. Lentiviral vectors were produced according to Protocol C described for Fig. 2a. DMEM lacking serum and chloroquine but supplemented with 0.3% HyQ Lipimate, 2 mM GlutaMAX and 100 U/ml penicillin and 100 μg/ml streptomycin was used.

    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 lentiviral transduction

    Addgene Lentivirus Production Protocol, Transient Transfection of 293T cells

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  6. For each lentiviral transduction, 0.25×10 6 NK cells per well were seeded in a 24-well plate BD Biosciences and mixed with an appropriate amount of virus supernatant in the presence of 8 μg/ml of protamine sulfate Sigma-Aldrich or polybrene Sigma-Aldrich in a final volume of no more than 1 ml.

    • Inhibition of Intracellular Antiviral Defense Mechanisms..
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    Why is Polybrene added to a transduction?} Polybrene also called Hexadimethrine bromide is a cationic agent that is thought to shield the negative surface charges of virus and cell, thus increasing transduction rates by decreasing repulsion. Depending on the cell type it may have a huge effect and can increase the titer 2-fold up to 10-fold. If kept for extended periods of time, chloroquine is toxic. The solution in the plates looks fuzzy, due to a very fine, dust-like precipitation of the transfection mixture. Note It is important to do all media changes with extreme gentleness, as the cells have been sensitized by the chloroquine, and can easily detach from the plate. How do you do transfection for lentivirus production. Lentiviral Transduction. For CaPO3, I use chloroquine, which inhibits DNA degradation by lysosomes by raising their pH - but this is.

     
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