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detaching cells but leaving matrix behind

detaching cells but leaving matrix behind - Protocols and Methods Forum

detaching cells but leaving matrix behind - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-21-2004, 12:52 PM
jg374@cam.ac.uk
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Default detaching cells but leaving matrix behind



I was given a protocol to detach some CHO cells from the flask so as to
leave the matrix behind, It was 15min at 37 with PBS(Ca/Mg free) + 5mM
EDTA and 5mM EGTA and proteinase inhibitors. However this solution totally
fails to detach more than 30% of the cells even if I leave it for 30 mins
and give the flask a good bashing. Any ideas how I can improve it's
efficiency without affecting the matrix, which I want to solubilize in
sample buffer?

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  #2  
Old 05-22-2004, 01:01 AM
Han
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Default detaching cells but leaving matrix behind

"[Only registered users see links. ].uk" <[Only registered users see links. ].ac.uk> wrote in
news:[Only registered users see links. ].c am.ac.uk:

I'd try putting them on ice after your treatment.


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Best regards
Han
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  #3  
Old 05-24-2004, 07:17 PM
Gys de Jongh
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Default detaching cells but leaving matrix behind


"[Only registered users see links. ].uk" <[Only registered users see links. ].ac.uk> wrote in message
news:[Only registered users see links. ].c am.ac.uk...
Hi,
1) Wash the cells twice with PBS
2) Remove the last PBS wash

Now we found the following three treatments will romove all the cells
leaving the extracellular matrix behind :

A) UV light for about 20 min
B) Heating in an air incubator at 60 degrees for 20 min
C) Store at minus 20 degrees overnight in the deepfreeze
one freeze/thaw cycle will kill all the cells

All three methods will give you a dish with the extracellular matrix and a few
attached , but dead , cells. The dead cells can be wased off with PBS before
seeding or will float in the medium at the first medium change after seeding.

C) works the best if cell growth on the matrix is compared
The dishes , wrapped in a plastic bag , can be stored in the deepfreeze for ever
I think. They still promoted cell growth after a couple of years

hth
Gys


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