Here is the medium composition which i used

RPMI-1640 medium supplemented with 10% FBS, 1 mM Sodium pyruvate, 1 % Non-essential amino acids, 10 ng/ml-Insulin

Test them for mycoplasma contamination, this may be one, of - of course several - reasons why your cells are in a poor condition.

Hi Anu Gopal,

As Sabine suggested, I would test for mycoplasma contamination and in the mean time try using a new batch of cells and observe if the problem persists. Please make sure that you use  ATCC -formulated Eagle's Minimum Essential Medium supplemented with  0.01 mg/ml

human recombinant insulin; fetal bovine serum to a

final concentration of 10%

Hope this helps!!!

thank u all....is it neccessary to maintain co2 percentage?

MCF-7 usually very viable cells. We was successfully used DMEM, DMEM/F-12 to maintain this cell line.

I guess problem could be in incorrect pH level. If you don't use CO2 incubator - it is a problem to maintain correct pH level in MEM (as long as it contain NaHCO3). Common level of CO2 must be 5 % since formulations of media made to balance pH in this conditions.

Try to collect media after incubation f.e. 24 hr and estimate pH level with pH metter (if media contain phenol red it could be tested by color changing: yellow-acid, pink-alkaline, brickred-neutral).

Also I agree with other - it could be ralated to mycoplasma contamination as well as to bad lot of FBS or long time of complete media storage (add L-glutamine at final concentration 2 mM).

Take attention to cell density, plastic properties etc.

what about other cells? Are they at good conditions?

i am also maintaining hela cells ..they are in good condition

Anu, it sounds like you have several issues here. Daniil is right that MCF-7 are robust cells that grow well in culture. MEM is designed to be used in a CO2 incubator. If you haven't got one, try using Leibovitz's L-15 media as it is designed to be used at atmospheric CO2. If cells are not growing sometimes this is due to the media not having Glutamine in the formulation - vendors often sell media without Gln, so check the formulation of the bottle you have. I would advise caution when culturing HeLa cells and another cell type in the same incubator. HeLa cells are very aggressive and have a history of taking over other cultures due to cross contamination. Good Luck! Ewan

Hi Anu, We generally use DMEM with 10% FBS in our lab to grow MCF-7 cells and so far did not face any growth related issues. I agree with Ewan that the choice of your media is problematic and also check for mycoplasma.

I faced similar problem. I used estrogen (Recommended in some literature )in medium but it is not working. I think MCF-7 cells grow very slow. After cell seeding, 2-3 days cells were in good condition then deteriorated.

I have been working on the MCF-7 cell line for 5 years, and one thing is for sure that this cell line is extremely sensitive, even for the media's PH change in which you can visually verify by the color, the recommended mediaon the ATCC website is : MEM, fetal bovine serum 10%, Sodium pyruvate 1mM, non essentials amino acids  0.1mM, insulin 10 µg/ml

In  any case my suggestion is to thaw new cells and start working on them , and try using fresh media, and do not leave the cells with same media for more than 3 days.