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MCF-7 CULTURING - (Nov/25/2010 )

I am working on MCF-7 cell line, during its culturing I am having troubles of contamination but i could not identify it. Please help
DESCRIPTION OF CONTAMINANT
The contaminant is in the form of clumps., plus the cell line shows a thin yellow film with granularity in consistency
The clumps or cell mass are visible by eyes

These contamination and cell clumping was not seen initially for 3-4 days. I have grown my cells in T-175 culture flasks

Please help

-Meera-

Can you post a picture?

It could be that your cell line is over confluent, as MCF-7 form into clumps that look like "colonies" when they are over-confluent.

-bob1-

bob1 on Fri Nov 26 00:07:35 2010 said:


Can you post a picture?

It could be that your cell line is over confluent, as MCF-7 form into clumps that look like "colonies" when they are over-confluent.


Thanks Bob for your reply

I have discarded the above cell lines as after 2-3 washings with antibiotic soltn in DMEM culture medium and plating in presence of antibiotic also showed the presence of bacteria ., i was so upset with the result that i never took the pics.

i have discardd the cell line and started culturing the new lot, but i am facing a problem., that these cell lines are unable to adhere to the culture flask as well as in the plates., these cells are floating and thus are unable to adhere. I have also tried to plate these cell in presence of gelatin coated plates (0.1%, but no such success was achieved.

pic is attached as an attachment

please do reply



I am sending you the pics of the cell where it increase in number and show good growth but is unable to adhere to the surface

-Meera-

Hmmm, MCF7 should attach reasonably well. Do you have insulin in the medium?

-bob1-

bob1 on Mon Nov 29 22:41:53 2010 said:


Hmmm, MCF7 should attach reasonably well. Do you have insulin in the medium?



Yes !!
The components used were:

DMEM with glutamine and 4.5 gm glucose and sodium pyruvate, 1.5 g/L sodium bicarbonat
FCS (10%)
Insulin 10 microgram /ml
30 micro Molar of Phenol red

The calculation for 10 microgram/ml which I did
The Insulin which i used was of 40 I.U. (of 10 ml volume) (used as a stock solution from Biocon)
As 1 I.U = 45.5 microgram Insulin
therefore 40 I.U has 18.20 microgram Insulin
thus to get 10 microgram/ml concentration for 50 ml of medium I added 2.74 ml of Insulin solution from stock to 50 ml of DMEM medium

Procedure for Preparaion of medium
Added DMEM powder, insulin stirred allowed it to dissolve, I also added 30 micro Molar of Phenol red then
Attained a pH of 7.4
Filtered the medium
and then added 10% FCS
used for subculturing


I read a paper"Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.
Y Berthois, J A Katzenellenbogen, and B S Katzenellenbogen
Proc Natl Acad Sci U S A. 1986 April; 83(8): 24962500"
quote "Phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells with an affinity 0.001% that of estradiol (Kd = 2 X 10(-5) M). It stimulates the proliferation of estrogen receptor-positive MCF-7 breast cancer cells in a dose-dependent manner but has no effect on the growth of estrogen receptor-negative MDA-MB-231 breast cancer cells
At the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media, thereby reducing the degree to which exogenous estrogen is able to stimulate responses." un quote


Please reply

-Meera-

Ahh! MCF-7 are ER positive, and phenol red is an estrogen analogue, which means that the receptor is internalised and can cause the cells to do some funny things. Try using phenol-red free medium, that should help the cells attach.

-bob1-

You should use phenol red-free medium for MCF-7 since it is a well-known ER positive cell.
However, phenol red itself does not inhibit cell growth or adherence. (actually it stimulates!)

I suggest you discard your stock, and use another one.

-hangun-

hangun on Wed Dec 1 09:55:19 2010 said:


You should use phenol red-free medium for MCF-7 since it is a well-known ER positive cell.
However, phenol red itself does not inhibit cell growth or adherence. (actually it stimulates!)

I suggest you discard your stock, and use another one.


The hard part of the problem is that I only have one stock of the above culture which earlier seem to grow well., these cells are round in appearance as shown in pic but never get flat as shown in MCF-7 pics seen in internet. Please help.
:( :(

-Meera-

Please do suggest some papers or a detailed protocol for the growth of MCF-7

-Meera-


DMEM with glutamine and 4.5 gm glucose and sodium pyruvate, 1.5 g/L sodium bicarbonat
FCS (10%)
Insulin 10 microgram /ml
30 micro Molar of Phenol red


Hi Meera

We use DMEM/Hams F12 1:1 with 5% charcoal-stripped FBS + 10 mg/mL Insulin for our MCF7 medium.
Also we know from personal experience that they grow best in 25 mL in T75 flasks. I never figured out why, but they somehow "like" that extra supernatant.

As others suggested phenol red should not be a problem for subcultivation (but I would use phenol red-free medium for estrogen receptor-related assays).

Hope that helps.

-Eimerian-