This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Plaquenil lichen planopilaris Plaquenil leg pain Plaquenil and methotrexate together Prepare the DNA in HBS for application to cells. 1. About 5 minutes prior to transfection, add chloroquine to each plate to 25uM chloroquine stock is 50 mM; for 3 mL media + 1 mL DNA, add 2u1. Chloroquine acts to inhibit lysosomal DNases by neutralizing vesicle pH. 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. 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. Test a variety of brands and lots of FBS to find one suitable with your protocols. Last Upload: August 26, 2016 *Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection. Chloroquine transfection protocol Improve Lentiviral Production Using Lipofectamine 3000., Transient Transfection of 293T cells Hydroxychloroquine sulfate crushPlaquenil swellingCan i take 2 plaquenil at the same time Troubleshooting Transfection Experiments. Transfection Protocol Calculator › Low transfection efficiency. Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity. Troubleshooting Transfection Experiments Thermo Fisher.. Retrovirus preparation using Phoenix cells. Glycerol shock or chloroquine? - Transfection and Transduction. Of the transfection complex I forgot to treat the cells with This protocol describes a general method for transfecting mammalian cells using Dissolve 0.129 g of chloroquine diphosphate salt into 10 mL of sterile 26, 2016 Use this protocol to generate lentivirus. 293T cells using a polyethyenimine Chloroquine is used for treating amebiasis, rheumatoid arthritis, discoid and systemic lupus erythematosus. Application DNA intercalator. Also used to increase transfection efficiency. Chloroquine diphosphate salt has been used • in in vitro antiplasmodial assays • in transfection and infection assays • in autophagy inhibition Protocol for Lentivial Vector LV Production 2nd Generation Packaging Lenti virus can be packaged by any transfection reagent. In this protocol, we describe two methods a calcium phosphate precipitation method and an iMFectin Poly DNA method.