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[Merrique]

Hey everyone,
I have been working with MCF 10A and MCF 12A for a while now. While the 10A are growing fine, the 12A are causing problems.
First they grew very slowly, so additionally to the recommended supplements and additives, I have adjusted the medium to high Glucose. They have been growing better since, yet now they have started to form domes ("raised" islands made up of 20-100 cells), even when they are not completely confluent. Before, when confluent, the mostly became smaller and denser or died.
Will this cause a problem for functional assays? Should I throw them away and thaw new cells? Or is this a normal phenomenon? The cells are Passage 13.

Medium:
DMEM/Ham's 12 (Biochrom)
+25ml Horse Serum
+6.25ml L-Glu (final conc. 2.5mM)
+2.5ml Sodium Pyruvate (final conc. 0.5mM)
+7.5ml Hepes (final conc. 1.5mM)
+100µl EGF
+250µl Hydrocortisone
+500µl Insulin
5ml PenStrep

These are according to ATCC's suggestions (especially the additives, for all other cells I usually ad 5ml of the 100x solution (Gibco))

Washing with Prewarmed PBS, Trypsin with 0.25% Trypsin (aspirated immediately), Ratios 1:2-1:4

I hope there's someone out there who can help me.
Best Wishes,
Anna

[zienpiggie]

I have no experience with the cell lines you are using, but if I were you I would thaw another batch and see if the problem is consistent. If they are, may be you could seed at higher density. may be you could also call your cell supplier to find out if they have similar complains about the cells.