The medium I use for cell culture of MCF7 includes DMEM medium, 10%FBS, 1% Glutamine, 1% ABS and 1% Na Pyruvate. I give the subculture for each generation every 3 or 4 days, with the concentration of 1:4 for next generation. However, for the subculture each time, after digestion of MCF7 with trypsin, I see the flocculent condition of cells, which means the cells gathering into cluster. Could I ask how to avoid this condition and how to improve the condition of MCF7 cell culture? Thanks very much.
There are certain cell lines that come off in sheets/clusters when they are detached using Trypsin. If your cells also reattach in patches it could be that you have not resuspended the cells well enough to get (more or less) a single cell suspension.
Try detaching your cells with trypsin, add medium and transfer into a 50 ml Falcon tube. Resuspend the cells a few times with a 1000 ul pipette (don't create too many bubbles as some cells don't like it) and see if that helps to get a more even distribution.
Additionally, if you put the freshly seeded cells into an incubator move the flask in a cross (a few times to the left and right and to front and back), that way you also get a more even distribution (some people circle it and that just centers all the cells in the middle).
I hope this was what you were looking for and it helps, good luck ;-)
Hi,
recently we had also difficulities with setting up a protocol for maintenance of MCF-7 cells. Finally, we ended up with protocol different than for the rest of our cell lines. In short: Subculture only once a week, detach cells with trypsin, then resuspend in DMEM, centrifuge gently (3mins), and then - resuspend them properly using syringe with needle! (That is probably the most important part for you). Usually we use resuspending with plastic pasteur pippette, but in this case needle works much better.
Then, transfer 0,5 million cells to new T-flask (25 cm2). Replace medium every 2-3 days.
We use regular DMEM + 10% FBS with phenol red.
I hope this will help.
Good luck!
Also check the concentration of glucose in your DMEM and the concentration of Estradiol in your serum, since MCF7 is ER positive.
you should try high glucose (4.5 g/l). You can buy a glucose stock solution for cell culture from Sigma and then supplement it.
The quality and concentration for Michigan Cancer Foundation - 7 (MCF-7) are--1 mL, 1 x 106 cells/mL in 70% DMEM, 20% FBS, 10% DMSO.When grown in vitro, the cell line is capable of forming domes and the epithelial like cells grow in monolayers. MCF-7 is an adherent cell lines which will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. Prior to the cell-lines should be sub cultured in other to prevent the culture dying. To successfully subculture MCF-7, they need to be brought into suspension. the degree of adhesion varies fron cell lines to cell lines but in the majority of cases, proteases e.g trypsin, are used to release the cells from the flask, the next thing in to triterate using using a sterile pastures pipet 5-7times depending on the degree of clumps ----------------aliquote appropriately and gently and shake to allow even distribution of cells in flask and trust me, you are safe of the trouble. Best result.
hi,
I agree with Christoph Vogel and would like you to suggest high glucose. Also, We have used DMEM, High glucose with sodium bicarbonate and 1x antibiotics (Pen and Strep) + 10% FBS and the cells were happy.
Hi again,
I also agree, we use hi-glucose DMEM + 10% FBS. No other supplements are needed for our MCF-7 cells.
I would NOT recommend using antibiotics for no reason (unless you want to expose cells with e.g. environmental sample). Reasons are many, for example antibiotics can mask contamination which already could have altered cell behavior, and could lead to generally poor cell culture practice. With no antibiotics you have clear "control" - cells are either healthy, or contaminated (as you will observe almost all contaminations next day).
My opinion - do not use antibiotics until it is necessary.
But if you need - gentamicin worked well for me.
Good luck.
I agree with Jakub.
No antibiotics if cell culture is clean in general.
Good luck
Hey, this was loong time ago, but maybe someone looks it up and is happy about other opinions.
So trypsinising more than 2/3 min definitely augments cells dying, rather giving them a claps and shoarten Trypsin incubation time
Also if just splitting and no counting necessary its better to not segregate the cells into single cell suspension but mix roughly and split them then quickly, they do not like to be seperated and splitt to much ...probably due to the epethelial origin ....so the cells like to always stay in contact ...
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