Chloroquine in transfection

Discussion in 'Hydroxychloroquine Sulfate' started by masterbek, 20-Mar-2020.

  1. tushk User

    Chloroquine in 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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    Chloroquine-resistant cells efflux chloroquine at 40 times the rate of chloroquine-sensitive cells; the related mutations trace back to transmembrane proteins of the digestive vacuole, including sets of critical mutations in the P. falciparum chloroquine resistance transporter PfCRT gene. The morning of the transfection day, replace the media with fresh DMEM without PSG and containing 10 uL of 25 mM chloroquine optional. Wait ~5h before going onto the next step. Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. 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.

    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 transfection

    Chloroquine diphosphate salt - Sigma-Aldrich, PEI Mediated Plasmid Transfection - Bridges Lab Protocols

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  6. Containing 100 µM chloroquine with-out serum were added to each well, followed by incubation for 90 min in 5% CO2 at 37°C. Cells were washed once in PBS and cultured in R10 for a total of 24 h from the start of transfec-tion. LIPOFECTIN Transfection For each transfection with LIPO-FECTIN, 1 × 106 cells were distributed in

    • Evaluation of Methods for Transient Transfection of a..
    • Addgene General Transfection.
    • Helper Dependent Protocol - Stanford University.

    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”. To “bubble”, use two mechanical pipeters Pipet-AidTM, one with a 1ml pipette to bubble air into the DNA-CaCl2 solution and the other to slowly drip the transfection buffer into the conical tube. The luciferase activity in the transfected cells was maximal when the transfection was performed for 3 or 4 h in the presence of 100 μMchloroquine. The luciferase activity was also enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does. Chloroquine diphosphate 52 mg Deionized, distilled H 2 O 1 mL Dissolve 52 mg of chloroquine diphosphate in 1 mL of deionized, distilled H 2 O. Sterilize the solution by passing it through a 0.22-µm filter. Store the filtrate in foil-wrapped tubes at −20°C.

     
  7. anriton Guest

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