9 replies to this topic

[jazmenia]

i'm facing a difficulty in growing MCF-10A. They are very very very slow. What should I do?

This is the recipe of the mdia I used:

DMEM=500ml
Horse Serum= 25ml
EGF= 20ng/ml
Hydrocortisone= 250ul
Insulin= 10ug/ml
My usual antibiotic=5ml

I noticed that some people add cholera toxin and pen/strep, does it really make a difference.

Edited by jazmenia, 23 June 2009 - 05:06 AM.

[bob1]

The ATCC has this to say about culturing MCF10A

ATCC said:

ATCC complete growth medium: The base medium for this cell line is MEBM, which is supplied as part of the MEGM Bullet Kit available from Clonetics Corporation, Catalog No. CC-3150. To make the complete growth medium, add the following components to the base medium: All MEGM SingleQuot additives that are supplied with the kit except the GA-1000 (BPE 13 mg/ml, 2 ml; hydrocortisone 0.5 mg/ml, 0.5 ml; hEGF 10 ug/ml, 0.5 ml; insulin 5 mg/ml, 0.5 ml); 100 ng/ml cholera toxin (sold separately).
Temperature: 37.0°C

[jazmenia]

thanks alot

[lynnb]

jazmenia, on Jun 23 2009, 01:38 PM, said:

i'm facing a difficulty in growing MCF-10A. They are very very very slow. What should I do?

This is the recipe of the mdia I used:

DMEM=500ml
Horse Serum= 25ml
EGF= 20ng/ml
Hydrocortisone= 250ul
Insulin= 10ug/ml
My usual antibiotic=5ml

I noticed that some people add cholera toxin and pen/strep, does it really make a difference.

(1) I grow MCF10A cells, but have never tried growing them without cholera toxin.
(2) I have tried growing them without insulin added and this makes very little dofference (suprisingly).
(3) Growing the cells without hydrocortisone did not work. Growing them without EGF did not work.

I grow the cells in:
DMEM/F12 base media
5% donor horse serum
EGF 20ng/mL final
insulin 10ug/mL final
hydrocortisone 100ug/mL final
cholera toxin 10ng/mL final

The doubling time is slightly over 24hrs when grown in this media combination.

You don't state the final concentration of hydrocortisone in your media and they do need this to grow. And as I said I haven't tried growing them without the cholera toxin.....
Hope that is useful.

Lynn

[debarshir]

Hello,
I am also growing MCF10A cells. I have few questions.
1. I do have DMEM media, don't have DMEM/F12..can I just grow this cell in DMEM?

2. How should I dilute the Cholera toxin and Hydrocortisone?

3. What is the doubling time of MCF10A cells?

Thanks

Deb

[lynnb]

debarshir, on Jul 15 2009, 09:42 PM, said:

Hello,
I am also growing MCF10A cells. I have few questions.
1. I do have DMEM media, don't have DMEM/F12..can I just grow this cell in DMEM?

2. How should I dilute the Cholera toxin and Hydrocortisone?

3. What is the doubling time of MCF10A cells?

Thanks

Deb

Deb-

1. I Have not tried to grow these cells in DMEM. I suspect it would work as long as you add the other media components that are required for these cells to grow.
2. I rehydrate both the solid hydrocortisone and the cholera toxin in sterile water.
I buy 100mg solid hydrocortisone and add 2mL sterile water. I place this on a roller to mix until it is fully rehydrated. Then, in a tissue culture hood, I use a 5mL syringe and a 0.2uM filter tip to filter the redissolved hydrocortisone. I make 1mL aliqouts (the amount to add to 500mL media) in eppendorf tubes and freeze these at -20C.
I buy 1mg solid cholera toxin and add 1mL sterile water and mix it well. This is stored at +4C.
3. In my experience the doubling time of MCF10A cells is 24 to 32 hours, with later passages of these cells growing at the slower end of that range.

lynn

Edited by lynnb, 17 July 2009 - 09:48 PM.

[debarshir]

Thanks Lynn for your help. I am starting to grow the cells this week, and will let you know if I face any problems culturing them.

Thanks again

Debarshi

[lynnb]

debarshir, on Jul 21 2009, 04:26 PM, said:

Thanks Lynn for your help. I am starting to grow the cells this week, and will let you know if I face any problems culturing them.

Thanks again

Debarshi

D-

I would point out that I have found these cells to be VERY ADHERENT to the cell culture flasks.
The adherence increases with cell confluency, in other words if you let the cells get over confluent you might never get them off the flask to split them. Even without over confluency I still find these cells require 7 - 8 minutes of trypsinization at 37C with a good whack to the flask to get the cells off the plastic.
This is good I hope
lynn

[Laurence]

Now I am also facing these questions.This post helps me a lot.Thank you very much.

[hafezparast]

lynnb, on Jul 22 2009, 01:58 AM, said:

debarshir, on Jul 21 2009, 04:26 PM, said:

Thanks Lynn for your help. I am starting to grow the cells this week, and will let you know if I face any problems culturing them.

Thanks again

Debarshi

D-

I would point out that I have found these cells to be VERY ADHERENT to the cell culture flasks.
The adherence increases with cell confluency, in other words if you let the cells get over confluent you might never get them off the flask to split them. Even without over confluency I still find these cells require 7 - 8 minutes of trypsinization at 37C with a good whack to the flask to get the cells off the plastic.
This is good I hope
lynn

I read somewhere that complete tryosinization may takes up to 45 mins!!! then be paitiont!!! but you shouldn't use more trypsin because it increase the cell tendency to make clumps. add 2ml and pure out but a film layer of trypsin and check them every 5 min after first 10 min. it sounds odd but read this

https://brugge.med.harvard.edu/Protocols/Ca...atrigel.doc.pdf