Chloroquine retroviral transfection

Discussion in 'Canada Drug' started by MastFlamastt, 27-Feb-2020.

  1. Alex35 New Member

    Chloroquine retroviral 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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    Hours after transfection reaction has been applied to cells, gently remove media and replace with 10% glycerol or DMSO for 2-3 minutes. Remove and feed with complete media various references. *1 hour before transfection, replace cDMEM with cDMEM + 25 mM chloroquine. Add transfection reaction to this media, and incubate o/n JoVE. 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”. Ing for transfection. In general, the cells should be plated at a density so that they are 95-100% confluent at 24 hours post-transfection. * The addition of chloroquine to the medium appears to increase retroviral titer by approximately two fold. This effect is presumably due to the lysosomal neutralizing activity of the chloroquine.

    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 retroviral transfection

    Retrovirus preparation using Phoenix cells, Transient Transfection of 293T cells

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  6. In this study, the effect of chloroquine on transduction and associated toxicity of baboon CD34 cells and a variety of cell lines was studied. Materials and Methods. Cell lines. To study the effects of chloroquine on retroviral transduction of cell lines, three cell lines were chosen Hela cells, D17 and 208 F cells.

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    Retrovirus Production and Infection – de Lange Lab 3 Retroviral Infection Day 0 Plate cells at a density such that they are growing well for the entire duration of infection and will be confluent at the end of infection Day 3, How do you do transfection for lentivirus production. For CaPO3, I use chloroquine, which inhibits DNA degradation by lysosomes by raising their pH - but this is toxic to the cell, so media. Mammalian cell transfection is a technique commonly used to express exogenous DNA or RNA in a host cell line for example, for generating RNAi probes. There are many different ways to transfect mammalian cells, depending on the cell line characteristics, desired effect, and downstream applications.

     
  7. Katrin Baukina Well-Known Member

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  8. Vodonaeva Moderator

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  9. maxsz Well-Known Member

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