I'm growing keratinocytes and they aren't sticking to the bottle, they've been suspended for three weeks. I use DMEM with 10% FBS.
Hi Talita,
Assuming you are using standard tissue culture flasks and not suspension culture flasks (which have a hydrophobic surface preventing cell attachment), there is a problem with your cells. HaCats should grow in DMEM/10%FBS. The first thing I would do is a viability stain with Trypan blue to assess the percentage of live cells in your culture. If there are still live cells, you can try and coat a new flask with a coating matrix (e.g. from Thermo Fisher R011K) before seeding your cells. This should facilitate cell attachment. However, if you have a stock of frozen HaCat cells, you could save some time by thawing another vial, assuming that cell viability of the fresh cells is high. If you see the same thing with the new cells, you will need to get a new batch of HaCat cells. I can put you in contact with a lab if needed.
Hope this is helpful. Good luck
Hi Talita,
According to my experience, healthy HaCat cells robustly attach to any type of non-hydrophobic plactic flack/dish. Besides the viability check, you may also look for microbal contamination, that alters cell's attachment properties, simply by observing moving dots under microscope.
Hi,
check medium (type of DMEM-glucose-glutamine,NaHCO3 contents, type of FBS-heat inactivation and antibiotics)
check culture flask (culture flasks have some different surface caracteristics like TC treated, ultra-low attachment, cellbindcellbind treatment, etc. and a vent cap, plug seal cap)
check thawing process,
and try to remember freezing process (freezing medium and %DMSO?). Or storage (if you kept at -80C for a long time probably they are not alive).
We experienced this, if the passage number was quite high - the HaCaTs will not grow, or takes very long.
They should be stored in liquid nitrogen and not -80.
When thawing these cells - make sure you add the cells slowly into low warm media (literally drop by drop) to ensure the cells stay in "close contact" - this will result in the cells to grow faster - this is what we experienced.
I hope my advice will help your situation.
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