I am working with MCF 7 cell line. I ordered the cell line from NCCS Pune. I received a very high confluent flask. After a day, I splitted the cells. I am using MEM w/Earle's salts, 2mM L-Glutamine, 1mM Sodium pyruvate, NEAA and 1.5 gms per litre Sodium bicarbonate with 10% FBS and antibiotics. A day after the splitting, I observed that all the cells were detached and floating. I changed the media and the flask again but next day the cells were in same condition ? They are not adhering in the flask and a lot of cells are dead. What should I do ?
Hello Sakshi Goswami
When cells are approximately 80% confluent, they should still be in the log phase of growth and will require sub-culturing to ensure proper growth and health of the cells. When cells become over confluent, as you mentioned that they were too confluent, they will begin to die off and may not recover.
Did you check the cell viability percent during sub culturing? Subculture the cells in smaller culture flask (T-25) and give them some time to adhere. Usually MCF-7 cells adhere to the substratum after 8-10 hours. Otherwise, you may have to contact NCCS and request them for another flask if MCF-7 cells do not recover.
Best.
Hello Sakshi Goswami
According to ATCC, you need to add 0.01mg/ml (10mg/L EMEM) Human recombinant insulin. Try culturing the cells by supplementing the medium with Human recombinant insulin, if the problem persists, contact NCCS.
Hello Sakshi,
Some questions/suggestions:
What is the CO2-level of your incubator? Does it match the bicarbonate concentration to maintain a pH of 7.4? And what is the pH of the culture medium at the start and after some hours in the incubator? If cells are exposed to a very alkaline (ca. pH 8) for longer than approx 30 min, they may not survive (Does the medium contain a pH phenol red as an optical pH indicator?)
MCF-7 are usually cultured with the enriched version of MEM, i.e. DMEM, with 10% FCS - requiring no further additions (ideally also no antibiotics if your're careful).
Hope this may save your MCF-7 cells.
Hi
There are so many troubleshoots that can be done here
To start with I would Certainly agree with what others have suggested and my point of view is to check the CO2 levels as Angela M Otto has suggested.
Are you using the flask from the same manufacturer in which you originally cultured the cells? If the flask is from another brand then you might want to check that up. Another point is that check for the presence of vents on the flask.
What were the concentration and duration of trypsin treatment, speed of spinning the cells during their passage, and the seeding volume/split ratio?
Can you recall the cell number if you did a viability assay (trypan blue) after spinning down the cells?
What is the passage number of the cells?
What are the conditions of the incubator (Co2 as mentioned and temperature)?
Are you growing any other cells besides MCF-7? How about their health?
Other than that MCF7 can be easily cultured in DMEM (+10% FBS; 1% Antibiotic) and you can try to culture in DMEM
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