I am new in the field of transfection and I'm having a lot of problems in setting up the protocol with LIPOPLEXES in order to compare data previously obtained using Viromer Red protocol on THP-1 cell line. The company from which we have the lipoplexes send me a protocol but they did not specify how many cell I have to transfect but only the ratio between RNA and lipoplexes. They also gave me a number about DODMA Lipid concentration that is 2,68nM. What this number stands for? Which information are missing?What I need to know to set up the protocol??Help me please :-(
1. If you are comparing previous methodology, then use the cell seeding that was used in Viromer protocol for THP-1 cells. In case you cant find that, then usually for a 96 well plate, HeLa cells its 5000 cells/well and you can scale upwards to 24 or 12 well plates.
2. Lipoplexes would be a mixture of DODMA( 1,2-dioleyloxy-3-dimethylaminopropane ), PEG-DSPE, DSPC and cholesterol at for example molar ratio of 50:1.5:10:38.5 respectively. So the number you have 2.68nM is thhe DODMA stock concentration (although from what you describe you must have bought prepared Lipoplexes in that case never mind this number). Since they have provided you with a protocol for siRNA (I am guessing thats what you are using) to Lipoplex ratio, it should be straight forward.
3. If you are not sure of the well-plate format, here is a bit of an indication, doi: [10.2147/IJN.S136426], materials and methods- section- 'Immune simulation'
Cheers
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