adherant cells

Nicholas King (nickk@med.su.oz.au)
Fri, 13 Jun 1997 13:14:48 +1100

Morgan, we routinely do this in HUVECS: we use very low concentrations of
Trypsin which we have shown do not lower the detectability of the adhesion
molecules we look for (indeed we look for upregulation). You can show this
for yourself: just mechanically remove the cells in a non confluent culture
and compare them with trypsinised cells by FCM. We look at E-selectin,
P-selectin, ICAM-1, VCAM-1, MHC-I and II.

The tricks are simply
-to wash thoroughly (at least 2x) with PBS first to remove culture medium
-to be strict about the time and temp which you allow exposure to trypsin:
usually <3mins, at RTemp <24 Celsius in our hands
-we use 0.05% or 0.02% in PBS

If you need any more info, email me.

regards,

Nick

>From: ans033@abdn.ac.uk
>Subject: adherant cells
>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>Date: Thu, 12 Jun 1997 15:57:43 +0100 (BST)
>X-Mailer: ELM [version 2.4 PL21]
>X-PMFLAGS: 33554560 0
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>
>Could anyone suggest a method of removing adherant cell lines from culture
>vessels which does not remove surface adhesion molecules? At present i am
>studying surface cell adhesion molecules expression on human umbilical vein
>endothelial cells using flow cytometry.
>i would appreciate any suggestions.
>thanks
>Morgan
>
>

Nicholas J.C. King, M.B. Ch.B., Ph.D.

Department of Pathology
Blackburn Building, D06
University of Sydney
New South Wales 2006
Australia.

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