Chloroquine pretreatment before transfection

Discussion in 'Aralen' started by pollentia, 15-Mar-2020.

  1. .brrou 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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    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. For 293 cells the medium was replaced by fresh medium without applying a shock. The cells were then incubated for 1–6 days before the supernatant was harvested and analyzed by ELISA. Transfection efficiency was determined by staining β-galactosidase expressing cells with X-Gal after 24 h. Stable transfection for CHO cells Pretreatment of cells with free colchicine before addition of lipoplexes increased transgene expression both in the presence and in the absence of serum. Free vinblastine had similar effects; however, vinblastine was more effective ∼30-fold maximal stimulation when incorporated into the lipoplexes.

    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

    Medicines for the Prevention of Malaria While Traveling., Transfecting Mammalian Cells Optimization of Critical Parameters.

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  4. Chloroquine is an inhibitor of the lysosomal degradation of the DNA which is taken up by the cells, so as leelee said, transfection should have been successful, albeit at a slightly lower level than if you would have added the chloroquine. In my normal transfections, I never add chloroquine and get high efficiencies nevertheless.

    • Chloroquine use in transfection. - Tissue and Cell Culture.
    • Effects of Microtubule-Depolymerizing Agents on the Transfection of..
    • Transfection Guide Overview of Transfection Methods Promega.

    FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to 56 ℃ for 30 minutes. 25 mM chloroquine diphosphate Dissolve 0.129 g of chloroquine diphosphate salt in 10 mL of sterile water. Filter sterilize through a 0.22 μm filter. Aliquot 50-100 μL and store at -20 ℃. In the current study, we observed that pretreatment of COS7 and HEK293 cells with these lysosomotropic agents, chloroquine 100 μM and ammonium chloride 10 mM, for 4 hours prior to electrotransfection insignificantly altered eTE Fig 7a and 7b. The pretreatment was required because the lysosomotropic agents need to be in lysosomes before. Chloroquine This drug is taken once a week, starting about 1 to 2 weeks before your trip and continuing for 4 weeks after. But chloroquine is rarely used anymore, because it no longer works.

     
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    Chloroquine has long been used in the treatment or prevention of malaria from Plasmodium vivax, P. malariae, excluding the malaria parasite Plasmodium falciparum, for it started to develop widespread resistance to it. Interactions between Travel Vaccines & Drugs - Chapter 2. Interactions between Ciprofloxacin Mixture Oral and qt. Ciprofloxacin mixture oral and chloroquine Drug.
     
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    Addgene General Transfection 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.

    Chloroquine ameliorates carbon tetrachloride-induced acute.
     
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