Hi Prem

As above we use DMEM, but with 5% FBS (and no antibiotics or insulin). To be honest pretty much any growth medium will work with these cells. As for the antibiotics and insulin - they are unnecessary. Insulin has been included in many mammary cultures for mainly historic reasons. If you do a 'side-by-side' comparison using insulin vs no insulin, you will see no difference (as we did). Also, with regards to trypsinisation, we routinely use 0.05% Trypsin-EDTA and add 2 mL for a T75 for 2-3 mins then neutralise with full media. That has always worked well for us.

Good luck with your experiments.

@João: Yes phenol red has significant agonist activity on ER, and hence it will promote MCF7 growth - which is good unless you want to see E2-induced growth, in which case you'll need to use phenol red-free medium with charcoal stripped FCS. Also, your observations on Tamoxifen's mode of action are correct. In full media (media with phenol red and non-charcoal stripped FCS) you will predominantly see a growth arrest from Tam treatment. In phenol red-free media using charcoal stripped FCS you will see more death, but you will still see a significant amount of cell cycle arrest.

MCF 7 Cells' growth medium is DMEM + %10 FBS.

Monolayer culture.

Additionally what you want to learn ?

@ Dilek .. Please let me know whether it can cultured in liquid medium or on the solid surface ...I am bit confused. as i have read it is an adherent cell line. AND also suggest me what should be the concentration of trypsin and Edta for passaging .(whether both trypsin and edta used separately or is mixed form and what should be the final concentration if each of them is mixed together?

This link should have everything you need to know:

http://mcf7.com/mcf7_cell_culture.html

I culture in DMEM + 10%FBS, 1% pen strep, and 10ug/mL insulin. We use 0.25% trypsin (0.53mM EDTA) that is premixed and commercially available from Invitrogen (Gibco).

I also culture in DMEM + 10%FBS, although I do not use pen/strep or insulin. But, recently we have been discussing about insulin, phenol red, sodium pyruvate and removing steroid from the serum with activated charcoal. There are paper about the low estrogenic effect of phenol red. Can anyone explain why insulin is recommended? I have been treating my MCF-7 with estrogen and tamoxifen and they are not proliferating in the presence of estrogen neither dying in the presence of tamoxifen...

Cell Culture

MCF-7 Culturing Protocol:

Use Eagle's MEM, supplemented with 10% FBS, 1% penicillin/streptomycin. Can also add non essential amino acids (0.1 mM), Insulin (10ug/mL) and Sodium pyruvate (1mM). Add 10nM estrogen to media for a 3-4x increase in cell numbers. Maintain temperature at 37°C in humidified, concentrated CO2 (5%) atmosphere.

Once MCF-7 cells reach approximately 90% confluence on plates, remove media and passage cells by rinsing with 1xPBS twice.

Add 2-3 mL of warm (37°C) 0.25% Trypsin- 0.53 mM EDTA solution to cells to disperse cell layer. Observe under an inverted microscope. Dispersal should happen between 5 and 15 minutes. If cells are not detaching properly, place flask back in 37°C incubation chamber. Do not incubate for more than 3 minutes or so. Note: Do not agitate the chills during dispersal, either by hitting or shaking the flask. This may cause clumping as the cells detach.

Once MCF-7 cell layer is dispersed (3min at 37°C) deactivate Trypsin by adding 5 ml cells/Trypsin-EDTA to 10mL of complete growth medium (see step 1) in sterile tube. Aspirate cells by gently pipetting

Centrifuge cells in growth medium for 5 minutes at 125x g-force.

Remove trypsin/growth medium suspension from tube.

Resuspend pellet (MCF-7 cells) in 10 mL fresh growth medium (see step 1)

Plate 1 mL of suspension to each new plate containing 9 mL original growth medium (see step 1), and incubate at 37°C in humidified 5% CO2 atmosphere.

A 1:3 or 1:6 subcultivation ratio is reccommended

Hi Prem

As above we use DMEM, but with 5% FBS (and no antibiotics or insulin). To be honest pretty much any growth medium will work with these cells. As for the antibiotics and insulin - they are unnecessary. Insulin has been included in many mammary cultures for mainly historic reasons. If you do a 'side-by-side' comparison using insulin vs no insulin, you will see no difference (as we did). Also, with regards to trypsinisation, we routinely use 0.05% Trypsin-EDTA and add 2 mL for a T75 for 2-3 mins then neutralise with full media. That has always worked well for us.

Good luck with your experiments.

@João: Yes phenol red has significant agonist activity on ER, and hence it will promote MCF7 growth - which is good unless you want to see E2-induced growth, in which case you'll need to use phenol red-free medium with charcoal stripped FCS. Also, your observations on Tamoxifen's mode of action are correct. In full media (media with phenol red and non-charcoal stripped FCS) you will predominantly see a growth arrest from Tam treatment. In phenol red-free media using charcoal stripped FCS you will see more death, but you will still see a significant amount of cell cycle arrest.

Why insulin:

http://www.fefchemicals.com/biopharm/scientific-information/articles/how-does-the-insulin-molecule-work-in-mammalian-cell-culture/

What if we culture it in MCDB 131 medium?

Thanks for the answers, Ranadevan, Alasdair and Berrin. @BerrinSaracoglu The link you posted is correct, but is not specific. Insulin general actions in mammalian cells does not explain why it should be recommended in the culture of MCF-7, since insulin is not a general recommendation. In the particular case of MCF-7, I have discussed with a few colleagues from Rio de Janeiro University and it seems to be a consensus observation that insulin is not necessary if you supplement with 2% glutamin in a phenol red DMEM low glucose. If your culture is a high glucose DMEM with low glutamin, insulin might make some difference, but the culture is not stable. Labs which have used this glutamin high glucose low DMEM without insulin have been able to easily maintain their MCF-7 cultures. We believe there is a metabolism shift to a less glycolytic state. And it have been also observed in the cellular response to clotrimazole, a work from some colleagues in Rio PMID: 22347377 

can anybody tell me which kind of DMEM is better for MCF7 culture? high glucose or low glucose?

Thanks

Sohelia, I think DMEM/F12 to abolish the estradiol like effect of the phenol red :)

We are using RPMI with phenol red with additives of 10% FBS, insulin, P/S, non-essential aminoacids and L-glutamine. 

Hi,

MCF7 (ATCC® HTB-22™)

Organism: Homo sapiens, human  

Cell Type: epithelial 

Tissue: mammary gland, breast; derived from metastatic site: pleural effusion  

Disease: adenocarcinoma

https://www.lgcstandards-atcc.org/en/Products/All/HTB-22.aspx#generalinformation

Depending on your cells. You check and compare if they like RPMI1640 or DMEM high glucose. Choose the one in which cells are growing faster.

Hello! I found this conversation very helpful. I am going to start growing MCF-7 cells to study the effect of certain growth factors (different from estrogens) on the proliferation of MCF-7 and on the expression of certain genes. Would you think that high glucose DMEM with phenol red would be an appropriate medium?