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immunofluorescence + Flow Cytometry analysis


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6 replies to this topic

#1 Dall

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Posted 13 March 2009 - 10:54 AM

Dear all,

is it possible to de-attach fixed adherent cells which were immunostained, in any way, to analyse the cells for fluorescence content?
Or is the only possibility to stain the fixed cells in solution?

Kind Regards,

Dries.

#2 lotus

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Posted 13 March 2009 - 11:34 AM

Dear all,

is it possible to de-attach fixed adherent cells which were immunostained, in any way, to analyse the cells for fluorescence content?
Or is the only possibility to stain the fixed cells in solution?

Kind Regards,

Dries.


I would stain the cells in solution instead of trying to detach them after staining. The only ways to detach them (that I can think of right away) are trypsin, edta or some other protease/detergent treatment and all of them will damage the cell wall.....

#3 CKtong

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Posted 16 March 2009 - 02:25 AM

:)

Dear all,

is it possible to de-attach fixed adherent cells which were immunostained, in any way, to analyse the cells for fluorescence content?
Or is the only possibility to stain the fixed cells in solution?

Kind Regards,

Dries.


I would stain the cells in solution instead of trying to detach them after staining. The only ways to detach them (that I can think of right away) are trypsin, edta or some other protease/detergent treatment and all of them will damage the cell wall.....


I suggest you to stain after you have detached them. and not go stain surface marker after fix(you get false negative) fix them after stained. :lol:

#4 Dall

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Posted 17 March 2009 - 02:06 AM

Thanks already for the replies!

I agree to stain first for surface markers prior to fixing, but the target proteins are intracellular, so fixation and permeabilisation is necessary for this application.

Kind regards,

Dries.

#5 memo

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Posted 17 March 2009 - 06:45 AM

you can always spin/spot the cells again after fixation I quess?

#6 Dall

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Posted 26 March 2009 - 10:08 AM

you can always spin/spot the cells again after fixation I quess?

I see.. with a Cytospin. Interesting..

But do these cells remain attached tight enough to withstand the several washing steps?

Greetz,

Dries.

#7 tonix37

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Posted 27 March 2009 - 01:25 AM

hi,
this is an interesting link to Nunc Upcell Surface plates to obtain cells without trypsin use




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