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Detaching Breast Cancer cells (MCF-10A, HCC70) without trypsin- used for FACs

FACs Breast Cancer Cells MCF-10A

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4 replies to this topic

#1 xbritoe

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Posted 24 August 2012 - 12:43 PM

Hi, does anyone have any suggestions for removing breast cancer cells (MCF-10A and HCC70) from T-flask. These cell lines especially the MCF-10A are super super adherent and I don't want to use trypsin because I want to run FACs with these cells in order to see the expression of a specific membrane-bound receptor. I heard that using EDTA works, except I'm not sure what protocol I should use for these breast cancer cells since they are so sticky. Any suggestions or tips would be greatly appreciated!

Thanks so much.

#2 Curtis

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Posted 25 August 2012 - 12:16 AM

if you don't want to use chemicals then why do prefer EDTA to trypsin? I normally use a scraper. you need to scrape very slowly.

#3 ascacioc

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Posted 25 August 2012 - 11:09 AM

I am also for the scraper. But this would leave the cells that have lateral connections still attached and since you want to do FACS of single cells not clumps of cells... EDTA should also work for your cells since it would complex Ca needed for the cadherins to attach both for the lateral junctions and basal ones. But I am not sure about the concentration.

Andreea

#4 Curtis

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Posted 27 August 2012 - 08:57 AM

To add to Andreea's comment, you can scrape and then slowly pipet up and down to seperate cells from each other

#5 asli

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Posted 03 September 2012 - 11:04 PM

Hi.
Try checking answers to the similar question here
http://www.protocol-...osts/32711.html

I previously used PBS/EDTA(10mM) with colon cancer cell lines HCT-116, HT-29 and SW620. Worked just fine, I was surprised how cells could detach easily.

Best wishes.





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