First I would like to point out that, maybe simple typo, did you incubate in hood? Not in incubator? Other questions that I would ask would be: Did you coat transwell with anything or does it require any coating such as fibronectin? Are your Astrocytes healthy? Simple procedural suggestion is to pre-incubate transwell with medium used for growing Astrocytes or try using different cell densities?

Hi Kimiko, Yes I keep it in the hood for 3 hours. I wanted to keep it in the incubator but the 12-well plates aren't that deep as the insert has to be flipped to keep the astrocytes and if I put a cover the medium + cells flow.

The milipore inserts said they were tissue culture treated so no extracecellular matrix coating is required. I work with primary astrocytes and I usually coat the plates or flasks with poly-l-lysine but yest I did not coat the inserts.

If the medium that you use requires CO2 for pH adjustment (such as DMEM or RPMI), leaving them in hood for 3 - 4 hours will be a problem. I am not sure what kind of treatment was done to the transwell. But If you coat them with poly-l-lysine or similar substance, as long as it doesn't interfere with your experiment, cells will attach quicker. Good luck.

Kimiko