Previously, I used 0.05% Trypsin-EDTA (used 1.5-2.5 ml for T75 flask after one PBS wash) to detach them, it took 20-30 min incubation at 37 and 5% CO2. And the cells took very long time (12-15 days) to attach. As per ATCC instructions, complete culture medium was prepared. So, how can fix attachment and detachment issues of MCF10a cells? Please suggest me. Thank you.
Hi,
Did you use Trypsin inhibitor for inactivating trypsin? Also till the attachment issues are there, please keep a policy of centrifuging cell suspension so that residual trypsin is removed. Does it have serum in the medium? If so, yoy may try increasing serum percentage a bit if nothing works out for you.
All the best Shital Kumar Barman
Thank you Manish Gore . I did not use Trypsin inhibitor but I am using FBS.
Try using Trypsin inhibitor. ATCC mentions use of Trypsin inhibitor.
https://www.atcc.org/products/all/CRL-10317.aspx#culturemethod
Hello!
I'm using MCF10A cells since 10 years and I can confirm you that they are very difficult to detach.
One easy solution is to wash with PBS twice instead of once. This helps to reduce the residual FBS from the medium.
The other solution is to keep the PBS for 2-3 minutes (e.g. the second washing) in the incubator before trypsin.
The fact that they take a lot of time to attach could also be due to the substrate (in some cases a Fribronectin coating is necessary) or to the fact that they are suffering when you passage them (if you change the medium the day before passaging, their viability after passaging increases a lot).
Paolo Armando Gagliardi Thank for your kind suggestion. Yes you are right I solved detachment issuse by washing 2-3 times with PBS. Now the cells are not attaching and growing. I deactaivated and removed trypsin well. So could you please share the recepies of the complete culture medium used for MCF10A? Again thank you.
Shital Kumar Barman I'm glad that you solved your problems with this cell line!! For medium preparation I always use as a reference this paper:
Article Morphogenesis and oncogenesis of MCF-10A mammary epithelial ...
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