Chloroquine pretreatment before transfection

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  1. Lera 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. Dilute 100 mM chloroquine diphosphate 11,000 directly into the medium either before or after the addition of the calcium phosphate–DNA coprecipitate to the cells Step 3. 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

    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

    Chloroquine and amphipathic peptide helices show., Calcium phosphate–mediated transfection of eukaryotic cells Nature.

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  7. 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.

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    Pretreatment with 0.1, 1, and 10 μM chloroquine reduced infectivity by 28%, 53%, and 100%, respectively. Reproducible results were obtained from three independent experiments. Reproducible results were obtained from three independent experiments. 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. 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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