Chloroquine pretreatment before transfection

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  1. EnterDM Moderator

    Chloroquine pretreatment before 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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    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. In contrast, in the present study our results indicated that oxalate-induced mitochondrial edema and depolarization, as well as the oxidative injury of renal tubular cells, were markedly attenuated by chloroquine pretreatment but accelerated by rapamycin pretreatment, suggesting that autophagy activation has detrimental effects on oxalate-induced mitochondrial damage and oxidative injury in renal tubular epithelial cells. Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods.

    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 pretreatment before transfection

    Could chloroquine be used as an effective treatment for the novel., Autophagy inhibition attenuates hyperoxaluria-induced.

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  5. Rapamycin and chloroquine were dissolved in DMSO and PBS, respectively, as a stock solution of 10 mM, and were resuspended in saline before injection. All rats had free access to regular rat chow and were maintained at 25 °C and under a light-dark cycle during the experimental period.

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    Chloroquine has been used to increase transfection efficiency for calcium phosphate, DEAE-dextran, and some liposome-mediated gene delivery 63,65–68. However, effects of lysosomotropic agents on polymer and some lipid based methods of gene delivery are still controversial in the literature. Add the transfection mix dropwise being careful not to dislodge the cells. Incubate the cells for 18 h, or until the following morning. The following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete. Incubate the cells 24-48 h before checking for protein expression. Both adults and children should take one dose of chloroquine per week starting at least 1 week before. traveling to the area where malaria transmission occurs. They should take one dose per week while there, and for 4 consecutive weeks after leaving. The weekly dosage for adults is 300mg base 500mg salt.

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  8. wmedtota Moderator

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