Hello Everyone,

So this is my umpteenth attempt at transfecting cells in a 24 well plate, even though my chitosan-TPP nanoparticle which I have loaded with Cy3-tagged siRNA has an encapsulation efficiency of 98% and I have completed resuspended the hydrogen formed after ultracentrifugation in nuclease free water (as per recommendation of a previous paper. After cell transfection, I still see no signals in the chitosan loaded nanoparticles. I have added a lipofectamine control and this definitely works. For context I am transfecting porcine immortalized LLC-PK1 cells acquired from ATCC. SO I basically really urgently need to know:

1. What do I dilute my chitosan loaded nanoparticles after reconstituting them in PBS (or do I even need to do this again) in preparation for cell transfection? I used OPTIMEM last time to get the desired final concentration, but I could have equally calculated this from the stock I made...

2. Do I perform cell transfection with chitosan loaded nanoparticles in the presence of serum containing medium?

I have read so many papers and they refuse to comment clearly on what I have said before.. I simply just say chitosan nanoparticles were added to cells but how the nanoparticle suspension used for cell transfection was prepare right before cell transfection and into what cell containing medium it was placed is definitely not stated. I would really appreciate some advice.