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. Hydroxychloroquine risk Mechanism of hydroxychloroquine Chloroquine retroviral transfection Causes for plaquenil to stop working In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and assayed on day 2 or 3. Because the cells are added directly to the DNA, this process reduces the experimental time by one day and allows for high-throughput transfection of DNA in a plate or microarray format. Our laboratory uses PEI over other cell transfection reagents because of its low cost. This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-. Many cell lines can be transfected successfully with PEI but in our experience these two cell lines express the highest level of protein compared to other cells. Since the publication of the original method1, increases in efficiency have been achieved by including additional steps such as glycerol shock2 and/or chloroquine treatment3. This protocol is a modified version of a published method4, in which calcium phosphate–based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells were rigorously optimized. 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 Addgene Lentivirus Production Protocol, Transient Mammalian Cell Transfection with. Hydroxychloroquine tremorHydroxychloroquine plaquenil ra 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. - Protocol Online. Nature methods--磷酸钙转染_图文_百度文库. Improve Lentiviral Production Using Lipofectamine 3000.. 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 Transfection Experiment Transfection Efficacy Reagent Volume Tissue Culture Grade Chloroquine Diphosphate These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves. ProFection® Mammalian Transfection System—Calcium Phosphate E1200. The calcium phosphate transfection protocol is routinely used for both transient and stable transfection of a variety of cell types. The protocol is. Chloroquine, which is generally applied to cells with the DNA, appears to.