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cells attach but don't flatten out


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#1 sc_queen

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Posted 01 December 2010 - 08:09 PM

My MDA-231 and Hela cells attach/grow very well, but a lot of them don't flatten out and stay round. Has anyone encountered the same problem before with MDA231 or any other cell lines? Sorry, I have been asking too many questions here lately.. I'm new to cell culture and am the only one working with cells in our lab... Any input would be appreciated!

#2 bob1

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Posted 02 December 2010 - 02:08 PM

Both of these lines should attach extremely well and you should have little problem culturing them. What conditions are you keeping the cells under (medium, FCS%, CO2% etc.)?

Have you looked for contamination?

#3 sc_queen

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Posted 06 December 2010 - 08:22 PM

Both of these lines should attach extremely well and you should have little problem culturing them. What conditions are you keeping the cells under (medium, FCS%, CO2% etc.)?

Have you looked for contamination?


They attach well, but many of them don't flatten out and remain round. I use DMEM (4 mM L-glutamine, 1000 mg/L glucose, 1 mM sodium pyruvate, 3700 mg/L sodium bicarbonate) + 10% FBS, and culture them under 5% CO2. Maybe I should increase it to 10%, as theoretically, CO2% needs to be higher if medium contains more than 1500mg/L sodium bicarbonate, right? Also I noticed that the medium recommended by atcc is Leibovitz L-15 which does not contain either sodium bicarbonate or glucose. People in other labs seem to be using DMEM though. Maybe I should change it to Leibovitz L-15?

Thanks!

#4 bob1

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Posted 07 December 2010 - 03:52 PM

Those conditions look fine. You could try the 10% CO2, but it might not make much difference. How does the pH look if you are using phenol red?

What medium were the cells in when you got them? If they had been growing in DMEM, they should still be fine in it, but if they were in another medium, you should have weaned them on to DMEM slowly.

#5 sc_queen

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Posted 07 December 2010 - 07:39 PM

Those conditions look fine. You could try the 10% CO2, but it might not make much difference. How does the pH look if you are using phenol red?

What medium were the cells in when you got them? If they had been growing in DMEM, they should still be fine in it, but if they were in another medium, you should have weaned them on to DMEM slowly.


The pH looks fine. It turns to orange in about 3-4 days. I was told to use DMEM, as the cells had been growing in that medium. The cells are growing well. It's just that many of them don't flatten out. I will take a picture next time for you to take a look. Maybe I am just being paranoid.

Thanks for your help every time!

#6 sc_queen

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Posted 08 December 2010 - 01:40 PM

Hi Bob!

Here are the pictures. How do they look to you?

Attached Thumbnails

  • MDA-231_20101208.jpg
  • MDA-231_20101208_4x.jpg


#7 bob1

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Posted 09 December 2010 - 11:26 AM

How confluent were the cells before you seeded them? If they were too confluent, you could have converted the cell type to mesenchymal rather than epithelial, so they could behave differently.

#8 sc_queen

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Posted 09 December 2010 - 12:24 PM

How confluent were the cells before you seeded them? If they were too confluent, you could have converted the cell type to mesenchymal rather than epithelial, so they could behave differently.


So that means they don't look right... They were 80-90% confluent before I seeded them. Is there any way of converting them back to epithelial type??

Edited by sc_queen, 09 December 2010 - 12:25 PM.


#9 bob1

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Posted 10 December 2010 - 07:30 PM

It looks from your pictures that the cells are quite confluent even after your seeding, which could affect the phenotype. Try seeding at a lower concentration - it should revert the phenotype.




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