RE: Stimulating Human Bcells with CpG

hi

My guess is that 48 hrs is too early.  are there papers that you are following that show the kinetics of generating plasma cells in vitro after CpG ?  And do you really expect to generate memory cells in vitro with only CpG ? I would be interested, let me know if you have citations that show that, I will admit know mouse literature better than human.

Also, you might optimize your dose and kinetics of stimulation using a more proximal end-point,  like up-regulation of activation markers. and, use a positive control like LPS or anti-Ig,

=======================================================
Rachel M. Gerstein, Ph.D.
Associate Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX) 



-----Original Message-----
From: inesrolim@igc.gulbenkian.pt [mailto:inesrolim@igc.gulbenkian.pt]
Sent: Tue 5/6/2008 11:30 AM
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