Hello everyone,
I am working now on optimizing mammospheres culture condition for the MDA-MB231,MCF7 and 4T1 cell lines. My medium is a serum free medium DMEM/F12 supplemented with B27(2X), bFGF,EGF and Heparin.
After 5 days of culturing mammospheres most of them are dying.Thus,it is not possible to carry out a second passage.
Any suggestion please.
Thanks in advance.
Few questions before I understand your experiment.
(1) How do you know they are not surviving. Do you just see single cells floating? or there is another method you know they are not surviving.
(2) Does DMEM/F12 also contain glutamax? If not may be add it and see if it helps.
(3) MDA-231 spheres are irregular, whereas MCF7 spheres are golf ball like structures, it will be good if you can share pictures of your spheres that you see.
(4) Just to be clear you add 1 ml B27 in 50mL media?
Hi,
Can you share your protocol. How often you change the media? What is the source of media you are using? I am using #D2906 or #D6434 from sigma. Do check the viability of the mammospheres using trypan blue. MDA-MB 231 and MCF7 both form very nice mammospheres. You can also try increasing the EGF concentration. With the media components you share it should work.
Best
We also add insulin and pituitary extract into our sphere forming media. Certainly for MCF7, insulin is a potent survival factor (I'm not sure about the other lines). We use the "Single Quots" reagents from Lonza which provides all of the growth factor supplements. In our experience, most lines do not form true spheres but rather they grow as loose and irregular clumps of cells. MCF7 do from true spheres. I assume you are using low-adherent plates (HEMA-coated)?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line