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Detachment of very adherent cells for flow cytometry


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#1 robradford

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Posted 23 July 2009 - 04:40 AM

First of all, this is my 1st post so hello everyone!
I'm having a bit of a problem with my cell line. We are using a new renal epithelial cell line which differentiate very nicely. We are trying to do some flow cytometry, but one of the side effects of the differentiation process is that the cells lay down a pretty impressive basement membrane. So much so that 10x Trypsin and Accutase really don't have an effect after 30 minutes or so at 37 degrees! Does anybody know of an alternative method (aside from scraping) to the two that I've mentioned to remove the cells from the plastic? Any advice would be very much appreciated!
Thanks,
Rob

#2 little mouse

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Posted 23 July 2009 - 05:18 AM

you wash your cells with PBS w/o Ca and Mg before stripping? and it stills stick? wow.
do you use trypsin with EDTA? from invitrogen you can get trypsin 0.25% plus EDTA (normally we use 0.05% plus EDTA).

#3 robradford

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Posted 23 July 2009 - 05:29 AM

you wash your cells with PBS w/o Ca and Mg before stripping? and it stills stick? wow.
do you use trypsin with EDTA? from invitrogen you can get trypsin 0.25% plus EDTA (normally we use 0.05% plus EDTA).

These cells are incredible, they are washed in PBS without Ca and Mg and our trypsin is 10x trypsin (i.e. 0.5%)! There is no EDTA in our 10x trypsin, but the Accutase does have EDTA and it doesn't work. Would pre-washing with EDTA, and then adding trypsin maybe help?

#4 cancergeek

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Posted 23 July 2009 - 05:57 AM

You might want to try a system like this. http://www.nuncbrand...e.aspx?ID=11850. UpCell plates by nunc are plates which have surfaces thats charecteristics change depending on the temperature the plate is incubated at. At 37C the surface is hydrophobic and promotes cell adhesion. When cooled to 20C the surface becomes hydrophilic and the cells detach. No enzyme treatments needed. You'll have to triturate to break up clumps but it should work for your purposes and you can order free samples.

Edited by cancergeek, 23 July 2009 - 05:58 AM.


#5 robradford

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Posted 23 July 2009 - 06:24 AM

Thanks cancergeek, that seems like quite an elegant solution! I'll see if maybe I can order some of those plates and I'll let you know how it goes.

#6 little mouse

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Posted 23 July 2009 - 06:40 AM

i would try to add EDTA with the trypsin, but the product cancergeek is talking about seems very interesting. I didn't know it. (but my cells are detaching with EDTA, or trypsin or accutase)

#7 robradford

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Posted 23 July 2009 - 07:04 AM

i would try to add EDTA with the trypsin, but the product cancergeek is talking about seems very interesting. I didn't know it. (but my cells are detaching with EDTA, or trypsin or accutase)

It does seem like a really good product, I was working with a polymer like that for my undergrad thesis and was surprised that there wasn't a million and one different uses for it. This one is very elegant indeed! I've ordered the free sample plates but I will definitely try adding EDTA to my trypsin to see whether that works. Thanks for the help guys/girls. I'll let you know how it goes.

#8 SuMi

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Posted 24 July 2009 - 01:06 AM

I have used those plates and found that they were good. ThermoFisher will give you a discount if you want to order them as they are a new product for them.

However these plates are expensive and so what I do to detach my (very) adherent cells is remove the media, add 5mM EDTA for 15-20 mins, pipette up and down in each well and remove the EDTA and cells. Then add 10% FCS in PBS and wash each well again and add the wash to your cell mixture. This stops the cells from being so sticky. Then I just wash the EDTA off my cells. I had lots of problems detaching my cells and this method just pops the cells right off the plate.




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