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cells can't be lysed after crosslinking


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#1 signstem

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Posted 08 April 2010 - 05:36 AM

Dear Fridends

I'm trying to lyse my cells with RIPA buffer after crosslinking with formaldehyde (20min). But when I checked them under the microscope with trypan blue, they looked pretty bright and intact. Does anyone has an explanation of this weired thing? Thank you very much in advance

#2 HomeBrew

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Posted 08 April 2010 - 06:16 AM

Do the cells lyse in the RIPA buffer without formaldehyde treatment? Do they look markedly different under the microscope after treatment with RIPA buffer with and without prior formaldehyde treatment?

#3 macroman

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Posted 08 April 2010 - 12:21 PM

Hi,
Once treated with Formaldehyde the cells become relatively resistant to lysing agents when compared with untreated cells. You can try decreasing the time for formaldehyde treatment and sonicating the samples in RIPA buffer.

Hope this helps.

#4 bob1

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Posted 08 April 2010 - 04:37 PM

First of all.... your fixed cells are dead, there is no doubt about that! As far as I can make out, fixing the cells in formaldehyde results in retention of enough of the cell membrane structure so as to still exclude trypan blue.

#5 signstem

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Posted 09 April 2010 - 11:42 PM

Dear Fridends

I'm trying to lyse my cells with RIPA buffer after crosslinking with formaldehyde (20min). But when I checked them under the microscope with trypan blue, they looked pretty bright and intact. Does anyone has an explanation of this weired thing? Thank you very much in advance



Thanks guys for all your reply.
When I lyse the cells without fixation, I can clearly see the cytoplasm get lysed, although the nuclei remain intact. I think after fixation, the cells become very rigid and indeed, I have to try using sonication. Will update you the results soon.




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