RE: On ice or room temperature?

Albert,

Some Mabs simply work better at different temperatures. As an example, CCR5
binds better at 37C than at RT, therefore we commonly stain only with CCR5
for 20min@37C followed by all Mabs for 20min@RT. With 0.02% sodium azide in
the wash buffer, internalization is not a problem. When in doubt you should
test these side by side using different temps.

SP

Stephen P. Perfetto, MS.,MT. (ASCP)
Manager, Core Flow Cytometry Facility
Vaccine Research Center, NIH
Building 40
40 Convent Dr., Room 5507
Bethesda, MD 20892-3015


-----Original Message-----
From: Albert Tai [mailto:acktai@exelixis.com]
Sent: Thu 9/29/2005 6:28 PM
To: cyto-inbox
Subject: RE: On ice or room temperature?
Hi Dr. Mangada,
I think lower temp. may help to slow down the internalization of surface
receptor.  I have also tried using sodium azide at 0.1% which may achieve
similar results.  However, I have not compared the two methods side by side.
Regards,
Albert

-----Original Message-----
From: Maloy Mangada [mailto:Maloy.Mangada@acambis.com]
Sent: Thursday, September 29, 2005 5:12 AM
To: cyto-inbox
Subject: On ice or room temperature?




Hi All,



Just wondered if there is a consensus on whether cell surface staining (or
intracellular staining) is better at 4 deg C (on ice) or room temperature.
I've done all of my staining at 4 deg (per recommendation of the Ab
manufacturer) with good results but I've scanned the recent literature and
it seems that all methods papers I've looked at stains at RT.



Looking forward to your comments before amending my procedure!



Maloy Mangada, PhD

Scientist, Cell Biology

Acambis, Inc.,

38 Sidney St.,

Cambridge, MA 02139

Email: maloy.mangada@acambis.com

Phone: (617) 761-4288

FAX: (617) 494-1741




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