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. Hydroxychloroquine and thalassemia Can plaquenil be used in the treatment of multiple myeloma Ocular safety of hydroxychloroquine Chloroquine is a member of quinolone family and is a weak intercalating agent. 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 Add 22 l of 25mM chloroquine to each 15cm dish for 10cm plate 7.8 l of chloroquinei.e. final concentration = 25 M. The cells should be transfected 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”. 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. Add transfection reaction to this media, and incubate o/n JoVE. 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 retroviral transfection Transfection Methods Overview - Bio-Rad, Transient Transfection of 293T cells Hydroxychloroquine tablets usesTreatment of uncomplicated malaria chloroquineChloroquine resistant p falciparum Retrovirus Transfection Protocol Prepared by Berggren, Travis [email protected] Submitted April 24, 2012 Submitted by Berggren, Travis [email protected] from Salk Stem Cell Core in-house protocols Contributors Lutz, Margaret. Modesto, Veronica. Panopoulos, Athanasia. Affiliation The Salk Institute Introduction Retrovirus Transfection Protocol. Glycerol shock or chloroquine? - Transfection and Transduction. Retrovirus Production and Infection. FAQ Retroviral Expression and Packaging. Q What is required to make retrovirus. These cells require transfection of only an expression vector to produce retrovirus. Cotransfect the Plat-GP Retroviral Packaging Cell Line, which stably expresses MMLV Gag-Pol, with pCMV-VSVG and an expression vector. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. Day 3 48 hours post-transfection - infecting cells with retroviral supernatent. Pipette supernatant from transfected Phoenix cells into 15 mL tubes and centrifuge at 1500 rpm for 5 minutes to pellet cell debris. Filtering through 0.45 um filter removes cells as well.