RE: Stimulating Human Bcells with CpG

Hello Ines,

What are you measuring in relation to these subsets?
You should  be looking for changes in maturation, proliferative status and Ig secretion
if you want to measure CpG directed B cell subset effects


Gill Webster PhD
Chief Scientific Officer
Virionyx Corporation Ltd
P.O.Box 91806
Auckland Mail Centre
New Zealand.
Phone: +6496362513
Fax: +6496362501
email: g.webster@virionyx.com


-----Original Message-----
From: inesrolim@igc.gulbenkian.pt [mailto:inesrolim@igc.gulbenkian.pt] 
Sent: Wednesday, 7 May 2008 3:30 a.m.
To: cyto-inbox
Subject: Stimulating Human Bcells with CpG



 Dear all,

 I'm trying to stimulate human B cells with CpG ODN2006-G5.
 I stimulate PBMCs with 12uM CpG, during 48 hours. I'm analysing them at  FACSAria  and
I'm using the following staining to differentiate Memory B cells and  Plasma
 cells:

 CD19 - Pacific blue
 CD20 - APC Alexa Fluor 750
 CD38 - PE-Cy7
 CD138 - PE
 CD27 - APC
 IgD - Fitc

 Memory B cells phenotype: CD19+/CD20+/CD38+/CD138-/CD27+/IgD+
 Plasma cells phenotype:   CD19low/CD20-/CD38+++/CD138+/CD27hi/IgD-

 We didn't find any difference between stimulated and unstimulated cells.

 Do you know if CpG is adequate for Human B cell stimulation? Should I be  using higher
concentrations? Or am I looking at the wrong populations?


 Thank you,
 ines