I am currently working with MIN6 cells. I am finding it tough to keep them in culture for more than 4-5 passage. I am using DMEM (25mM glucose, 3.7 grams NAHCO3), 4mM glutamine, 1mM sodium pyruvate and 15% FBS. I am seeding 2.5 million cells in T25 flask. Kindly help?
Hello
I suggest one of this protocols :
In protocol A : These cells were cultured in DMEM with 25mM glucose,15% inactivated FBS , 79uM (micromollar) of 2-mercaptoethanol , 1% penicillin/streptomycin , 2mM glutamine , 100 µM 2ME ,15mM HEPES ,5% CO2
In protocol B : These cells were cultured in high glucose DMEM (Gibco) containing 10% (vol/vol) heat-inactivated FBS and 2-mercaptoethanol (3.5 μL for 1 L of medium). Cells were grown in 5% (vol/vol) CO2 at 37 °C and passed by trypsinization when they reached >80% confluence. The culture medium was changed every 2–3 day .
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
Good Luck
If possible, grow cells in different sera. MIN6 cells will crash in poor quality serum.
Hi there,
I am having problems with MIN 6 cell culture now. I had read so much in order to figure out my problems. So far, I still suffering from this. Could anyone give me any advice? My cells were looking great after thawing out in a T25 flask and the following subculture in a T75 flask for one generation. Things went wrong when I splited cells from a T75 flask into two T150 flasks (seed 5.7 million cells each flask). After four days of culturing, cells started looking very badly as 60% of cells detached.
My culture medium is DMEM (high glucose, Gibco) supplemented with 10% heat-inactivated FBS , 1% penicillin/streptomycin, 15 mM HEPES. Change medium every second day and cells are culturing in 5% (vol/vol) CO2 incubator at 37 °C.
Please kindly help.
Jessie, you are missing sodium pyruvate and beta-mercaptoethanol in your media. My media was DMEM (25 mM Glucose) supplemented with 10% FBS, 4 mM L-glutamine,1 mM sodium pyruvate, 10 mM HEPES, 50 microM beta-mercaptoethanol, and 1% Pen/Strep. Also, I had better luck growing my cells at 10% CO2 than 5%. Poor FBS will kill your cells. You may need to test different batches.
Typically, it is better if you expand into a similar size flask or dish at 1:3-1:6. Going from a T75 to two T175 may have decreased the density where your cells just simply didn't proliferate. I split the cells every seventh day. I changed the media on Day 3 and 6 after splitting them.
Thank you very much for your prompt reply Mario.
The DMEM I got contains 1 mM Na-Pyruvate but I don't add beta-ME in my medium as the people I learn MIN6 cell culture from never add beta-ME in their medium. I was thinking instead of adding beta-ME, they may avoid the accumulating oxidation of antioxidant pools by replacing fresh medium regularly (every second day). Do you think it is possible?
I would definitely try the medium you suggested with beta-ME and test with different batches of FBS to see if I can grow MIN6 cells back.
Thank you again for your suggestions.
I am observing a lot of background in my cell culture. I cannot figure out why. The background increases after 36 hours despite the aforementioned conditions by Saleh (protocol A) and even Mario(except for increase in Co2 to 10%). Could you please help me find my problem.
Why dont you add 2-mercaptoethanol in culture media? Min6 cells were different type of cell like mixed cell line with other pancreatic endocrine hormon. And what is your passage number?
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