Re: Detaching cells off TC plate after fixation

From: Albert Tai (acktai@exelixis.com)
Date: Tue Jan 28 2003 - 21:12:54 EST 

Hi Gill,

Unfortunately I did not get any response on my question.  However, I have explored different methods using formaldehyde, alcohols and detergents without any success.  Basically, after I fixed cells with formaldehyde and then permeablized with detergent or alcohol, cells will detach after application of trypsin.  But the morphology of the cell changed dramatically as seen in FSC vs. SSC and they looked like cell debris and yet would fluoresce.
Apparently, the trypsin was still at work even with addition of 10%FBS/PBS to neutralize trypsin action.  Any thoughts would be much appreciated.

Regards,

Albert Tai
Exelixis, Inc.
  ----- Original Message ----- 
  From: Webster, Dr Gill 
  To: Albert Tai 
  Sent: Tuesday, January 28, 2003 6:05 PM
  Subject: RE: Detaching cells off TC plate after fixation


  Hi ALbert
   
  what replies did you get to this question?
  thanks
   
  Gill Webster

  Head Of Flow Cytometry
  Senior Staff Scientist
  Genesis Research And Development
  1 Fox Street, PO Box 50
  Auckland
  New Zealand

  tel 0064 9 373 5600
  fax 0064 9 373 2189  

    -----Original Message-----
    From: Albert Tai [mailto:acktai@exelixis.com]
    Sent: Saturday, 21 December 2002 8:09 a.m.
    To: Cytometry Mailing List
    Subject: Detaching cells off TC plate after fixation


    Hi fellow flow-ers,

    Happy Holidays !  I would like to seek some advices from you for detaching fixed cells in 96 well TC plate(by formaldehyde and then permeabilized by MetOH) to run on BD multi-well autosampler.  Is it possible to lift the cells off the well after fixation ?  What would you recommend, trypsin, accutase or cell dissociation buffer to preserve surface receptor for Ab staining ?

    Thanks very much in advance for your help !

    Albert Tai

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