I've been having a hard time forming an endothelial monolayer with good tight junctions. The person who trained me on the culture of endothelial cells worked mostly with mouse BMEC, and he used ZO-1 to visualize tight junctions. He didn't get much of an internal signal of ZO-1 when he would do his staining, but I always get massive amounts of it (regardless of if I permeabilize the cells or not). I've attached images of my cells (hBMEC, passage 4), stained for ZO-1 & DAPI; the first image is 2 days of subculture, 2nd image is 4 days of culture (which didn't have as strong of a signal from ZO-1 & cells looked less confluent). Since subculturing the cells the first time, the quality of the cells only deteriorated, and idk why. I coat my dishes/flasks with fibronectin & seed at 5,000-10,000 cells/cm^2. Cells would be confluent in 2-3 days initially, but now it takes over a week! I started off using media from Lonza for microvascular endothelial cells & recently switched to one from Cell Biologics (where I bought the cells from) for human microvascular endothelial cells. Nothing I change up seems to help much :(

I've been struggling with them for awhile now...my current hypothesis is that I have a mycoplasma contamination in the lab, but nobody I talk to has any experience with that. The cells were beautiful when they first came in, but after about a week they started looking funky, growing slower, & detaching more frequently. The media never changed color or looked cloudy & I don't see anything weird floating in the media besides detached cells.

Any help would be much appreciated.