I need to transfect GFP vector and Luciferase vector plasmid DNA into the HL-60 floating cells. I will have to use X-tremeGENE HP DNA Transfection reagent from ROCHE. If anyone already experienced using this reagent, please share. How can I find a good protocol for the transfection of HL-60 cells using mentioned kit?
Hi, what analytical methods do you have available to check for your GFP and Luc expression? if you have a flow cytometer then I would recommend following the protocol provided with the reagent and transfect the appropriate amount of DNA (depending on the vessel you transact in) at several transaction reagent:DNA ratios- 5-1. I did this with a different suspension line and found a ratio of 3:1 to be optimal but it is cell line dependent.
For analysis, take a fraction of each transfection on days 1, 2 and 3 and assess viability, luck expression/cell (RLU/#cells) and GFP expression/transfection efficiency using flow cytometry.
If both of your plasmids use the same promoters you may run into an issue competition resulting in one being expressed more then the other. You may want to try 2 extra transfections- one with just GFP and one with just luciferase (make the total DNA transfected the same by using an empty vector plasmid. Also note that GFP expression itself is toxic in most cell lines so plan to transfect less GFP than Luc. you can assess its toxicity in the GFP alone transfection.
hope this helps you out.
Thank you Dr. Harbell. Yes, I'll use flow cytometry for analysis. Think your answer may help me out.
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