Hello, I am culturing HepG2 cells (passage 3) in EMEM with 10% FBS + 1% Pen/Strep and 1% L-gln. I am growing them currently in 10cm dishes, but soon plan to plate these cells into 6-well dishes (for RNA harvest-->qPCR) as well as 60mm dishes (harvest protein for western blot). My question is: what is the optimum seeding density of HepG2 cells particularly in 6-well dishes and 60mm dishes? I understand HepG2 cells tend to clump together, but I am looking to seed these cells at an appropriate density to get a decent monolayer without too much crowding or too sparsely.

To give you context, I will trypsinize these cells from the 10cm dishes, and while the cells are still in suspension (during the passaging process, before plating), I am adding an Adenoviral-vector to express shRNA to knock down my gene of interest in these cells (I have a good protocol for this adenoviral transduction). After plating the cells (in 6-well plates and 60mm dishes in 10% EMEM), I let the cells "rest" overnight and then the next day I come back and replace with 0.1% EMEM to growth arrest and allow the cells to incubate for ~48-96hrs before I harvest them for RNA (48hrs) or protein (96hrs). Therefore, I like to have an initial seeding density that will adequately create a monolayer and "remain" a monolayer for the next few days (because of the 0.1% growth arrest so they don't double over my time period). Can anyone give me advice on what seeding density to use for HepG2 cells on 6-well plates and 60mm dishes for this purpose?

Thanks!