MIN6 cells - (May/15/2007 )

Hallo, I'm a new member needing for help.
I'm trying to keep MIN6 cells in colture, but they seem to grow really too slowly...
Do you have experience with these cells?
I'm feeding them with DMEM with:
25mM glucose
15% inactivated FBS
1% penicillin/streptomycin
2mM glutamine
100 µM 2ME
15mM HEPES
5% CO2
according to some papers I found, but I don't have good results.
Do you have experience with these cells?


I hope you can help,
Lauretta

---

Hi, you need 79uM (micromollar) of 2-mercaptoethanol in the growing medium, too, otherwise the cells are likely to change morphology. They shoul look squarish. Also, Min6 cells tend to grow better if you seed more cells when you split. I'll say 1:4-1:6 or so. They like contact. Otherwise they will grow slowly and don't spread out

---

Thank you very much.
I'm actually already using b-mercaptoethanol (I refer as 2ME in my message), but i'll try to split them a little bit more concentrated.
They do not need special flask, do they?

---

Sorry about that, didn't look carefully enough. No, they don't need special flask. But they do grow quite slowly. When you said that you don't have good results, what exactly do you mean? Cells grow in clumps? Too slowly or not growing? Cells did not spread out? Cells look sick? Shape changes?

---

Well, i don't know which exactly should be the right shape, but about half of my cells are
round-shaped, and the other half are more fibroblast-like shaped, but with less
and shorter protrusions. They almost don't grow at all, I change the medium twice a week
but I can only split them once a week, 1:2.. They do not tend to form clumps.
Anyhow they do not seem sick or suffering..
Ps I thawed them 3 weeks ago.
How often do you change the medium?
Really thank you for helping..

---

Hmm, I wish you have picture. Fibroblast shape doesn't sound right. I only got that kind of fibroblast shape if I left them grow withour 2ME. When I seed 2.4x10^5 cells into 1 well of the 6-well plate, it takes 1.5-2 days for cells to start spread out (that is, you can see the shape), and 1 more day (3 days total) for them to reach about 30-40% confluent. About 3-4 days more and I can split cells again (they normally won't get to 100%). I split once a week if I split about one fourth to one sixth. I don't have to change medium often, only when I split cells or maybe once in between if I am feeling nice. Are you sure that your 2ME is good? Maybe need to ask someone who has good stuff, confirmed work.

---

I still don't have a picture unlickily.. So, which is the correct shape for Min6? Should they be rond cells? An other question, is it important to use the cells between passage 15 and 25? I'm reading it in almost every paper where they use MIN6. Do you or someone else know the reason?
Thanks.

---

The cells are broad, squarish looking. They tend to grow in clusters. Don't know much about the passage. For some cell lines I think the passage could be important but if you don't have the original tube, it may be hard to know which passage your cells are in anyway.

---

Hello, i still need your help..
do you think it' important to stimulate the cells in Hepes-balanced krebs-ringer bicarbonate (H/KRB) if i want to check MAPK activation?
Thank you in advance..

---

I have never done anything with signal transduction, so I am afraid that for this you will have to look for protocol in published papers or maybe text books. The thing is, Min6 cells are used mostly for secretory study, so I am not sure that not many will use it for MAPK activation. If you cannot find out in papers, then take one known worked protocol for MAPK activation and try yourself. Remember to have all controls so that you can troubleshoot (in case). Sorry can't be of much help.

---