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. Ocular side effects from plaquenil Plaquenil side effects fatigue Itchiness side effect of chloroquine Plaquenil toxicity in the eye MM chloroquine 1000x Sigma C6628 8 mg/mL polybrene/hexadimethrine bromide 1000x Sigma H9268 2M CaCl 2 2x HBS see recipe at end of protocol DNA retroviral vector Procedure Day 1 Phoenix cell preparation 1. Plate cells at 2.5×106 cells in 8 mL DMEM in a 10 cm dish one for each transfection to be done. System Mammalian cell transfection is a technique commonly used to express exogenous DNA or RNA in a host cell line for example, for generating RNAi probes. There are many different ways to transfect mammalian cells, depending on the cell line characteristics, desired effect, and downstream applications. 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. 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 Stable vs. Transient Transfection of Eukaryotic Cells., Principles and Mechanisms of Mammalian Cell Transfection. Hydroxychloroquine sulfate 200 tabsCan you take hydroxychloroquine sulfate if youre allergic to.sulfaPlaquenil assistance programPlaquenil treatment for vasculitis Ing for transfection. In general, the cells should be plated at a density so that they are 95-100% conﬂuent at 24 hours post-transfection. * The addition of chloroquine to the medium appears to increase retroviral titer by approximately two fold. This effect is presumably due to the lysosomal neutralizing activity of the chloroquine. PhoenixTM Retroviral Packaging Cell Lines Ampho and Eco. Helper Dependent Protocol - Stanford University. Transient Transfection of 293T cells. C H A P T E R 16 C ultivation and Retroviral Infection of Human Epidermal Keratinocytes Fiona M Watt, Simon Broad, and David M Prowse I. INTRODUCTION There are many techniques for culturing human epi- dermal keratinocytes, but the method described here is the one devised by Rheinwald and Green 1975. 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. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine.