LNCaP Cell Clusters - (Oct/26/2013 )

My LNCaP cells started growing in clusters/aggregates (cells grow on top of each other) rather than in a monolayer. I can actually see small bumps/clusters by eye in my flask. Does anyone know what I can do to make the cells grow in a monolayer again? 

When you plate them, do you let them settle before moving them to the incubator? When I started working with LNCaPs I was warned that you need to let the plate sit for 5-10 minutes immediately after you split them, otherwise they can get jostled and stick to one another before settling onto the plate. 

So I actually figured out the problem! The clusters/aggregates were a result of over-trypsinization. I was suggested (by a ResearchGate forum) to add a small amount of trypsin (1 mL) to the cells for a short amount of time (1 min), as soon as cells start to come off, I was instructed to add 10% FBS media and pipette the cells up and down 15-20x before splitting (1:2 or 1:3 ratio). This seemed to do the trick! Apparently, LNCaP cells are extremely sensitive to trypsin. After following the protocol to prevent over-trypsinization my cells started growing in a nice monolayer once again.