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 dosage sjogren& 39 Sulfasalazine and hydroxychloroquine side effects Plaquenil treatment for lupus When performing a transient transfection, you can choose between a standard or reverse transfection protocol. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and. Plate cells the night before to give 60-80% confluence at the day of transfection. 2. One hour prior to the transfection, change to medium containing 25µM chloroquine from x1000 stock in PBS, stored at -20°C. Volume should be 10 ml per dish. Chloroquine can be omitted, but increases efficiency about x2 3. The following are the protocols I've gathered *4-6 hours after transfection reaction has been applied to cells, gently remove media and replace with 10% glycerol or DMSO for 2-3 minutes. Remove and feed with complete media various references. *1 hour before transfection, replace cDMEM with cDMEM + 25 mM chloroquine. 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 reagent, Calcium Phosphate transfection of 293T cells Optic neuropathy due to plaquenilPlaquenil for chronic painPlaquenil toxicity autofluorescenceAralen rheumatoid arthritis If chloroquine is not used, it is unnecessary to change the medium prior to transfection. On some occasions, we have obtained slightly improved transfection efficiencies by adding the chloroquine to a 11 mixture of 293 conditioned media obtained from any of the 293-based cell lines and fresh GM. Pheonix 转染 protocol_百度文库. Glycerol shock or chloroquine? - Transfection and Transduction. Transient transfection into 293T cells by Jun Takagi, Jun 15.. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80°C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model. 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. 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.