Hi everyone,
I am currently working with the LUHMES cell line from ATCC and have run into a few issues.
First, the protocol for culturing them involves coating plates with 50 µg/mL poly-L-ornithine followed by 1 ug/mL Fibronectin, which works great. However, I want to plate them into 6-well plates containing acid-washed glass coverslips followed by coated with 100 µg/mL poly-L-ornithine and 10 ug/mL Fibronectin (which is suggested for everything other than routine maintenance of cells), but can't seem to get the coverslips to stay attached to the plate throughout the coating process and during cell culture.
The second thing is passaging with Trypsin. The protocol calls for 0.025% Trypsin-0.1 g/L EDTA solution, however, the cells do not easily detach from the plate even after 10 minutes of exposure and incubation. 0.05% doesn't work any better. I can get them to detach from the plate by gently knocking the side of the plate, but then they come off in sheets and are tough to resuspend/replate.
If anyone has any suggestions/ideas to fix either of these issues I would greatly appreciate it!
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