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Culturing HEBCs in 96 well-plates - (Oct/09/2012 )


I'm having a problem with primary human bronchial epithelial cells from Lonza. I've already used two different vials from the same batch and the problem remains. They grow happily in flasks with BEBM supplemented with BulletKit aliquots. However, when I transfer them to 96 well-plates (on passage 3) they start randomly dying after 2 or 3 days. The day after seeding they look absolutely fine but after that I constantly find random wells of dead cells... I don't understand what's going on... They don't seem contaminated. I have already tried 3500 and 10000 cells/well and got the same result. I've tested the first vial for mycoplasma contamination and it was negative...

Does anyone have any suggestions?


what type of 96-well plate did u use?
the u-bottom or the flat bottom?
I had the same problem when culturing my cells in the u bottom plate
but after i used the flat bottom one, the cells grew just fine..