questions about MCF-7 cells - (Jul/31/2006 )
I am going to use MCF-7 cell line. I have two questions which I need help on.
First, I noticed on the ATCC website that 0.01mg/ml bovine insulin should be included in the medium. However, the protocol I got from a colleague does not have the insulin. I am wondering what's the difference between inclusion or exclusion of insulin in the medium for MCF-7 cells?
Second, again on the ATCC website, the protocol suggests to centrifuge the cells after trypsinization. Do I need to centrifuge the cells every time I split them? What is the purpose of this centrifuge?
Hope those who have experience with MCF-7 cell line can give me some help. I would really appreciate it!
I dont use additional insulin. Cells are just fine.
Initially, we cultured MCF-7 cells in the ATCC recommended medium with the supplements (including insulin). I don't know why ATCC recommends centrifuge for subculturing, but we did not centrifuge the cells and did not experience any problems. Currently, we are growing MCF-7 cells in DMEM/F12 medium. There is no additional insulin and cells are okay.
We grow MCF-7 in "nomal" DMEM. (same to our 293cells and several other cell lines). and I was taught to split these cells in normal way (no centrifugation).. I think the cells are quite happy
Even if you don't use insulin for regular maintainance that's fine bcoz you are using serum in the medium. Serum is not completely defined supplement. But if you are doing some kind of assay try to add insulin and see whether it makes any difference to your assay results.
Centrifugation removes the trypsin as you resuspend cells in fresh medium. Having said that when you add complete medium containing serum for inactivation of trypsin, that is more than enough to inactivate the trypsin. So in essence you need not centrifuge your cells everytime. You can do so once in a while.
Hope it helps.
I use RPMI with 10% heat inactivated FBS to culture my MCF-7s and they grow like weeds. Another person i work with uses insulin for her WS8 cells (subclone of MCF-7). The insulin is used to help with the clumping (as told to her by Fox Chase... where she gets the cells from).
i've used media with and without insulin. cells seem perfectly happy either way.
my cells, though do like to clump regardless if there is insulin in there or not... syringing them is essential.
don't bother to centrifuge them after typsinisng them. they're very happy.
We use DMEM FBS 10% with no insulin.
We detach them with PBS/EDTA and no tripsin, but we do centrifuge them don't ask me why, it's just the protocol it has been used for centuries!!
...by the way... they do clump A LOT!
Why do we use heat inactivated FBS? What is the purpose?