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 and fibromyalgia Aralen anime Chloroquine prevention malaria 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. Prior to adding complex, remove 1 mL of medium from each well, leaving a total of 1 mL in each well. Add 500 μL of lipid–DNA complex to each well, taking care to dispense liquid against the well wall to avoid disrupting cells. Gently agitate plate to evenly distribute. Incubate plate for 6 hours at 37°C, 5% CO 2. The following protocol is for 10 cm dish medium 10ml. If you use 6-well or 12-well plates, total volume of the medium should be 3ml and 1.5ml, respectively, and decrease the amount of each reagent accordingly. 1. Plate cells the night before to give 60-70% confluence at the day of transfection. 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 Troubleshooting Transfection Experiments Thermo Fisher., Improve Lentiviral Production Using Lipofectamine 3000. Neostigmine chloroquine phosphate Test a variety of brands and lots of FBS to find one suitable with your protocols. 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. Addgene Lentivirus Production Protocol. Transient transfection into 293T cells by Jun Takagi, Jun 15.. Questions with answers in CHLOROQUINE Science topic. 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. This protocol is a modified version of a published method 4, in which calcium phosphate–based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney. 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.