RE: Stimulating Human Bcells with CpG

Ines,

48 hours is too short a period of time to see B cell division after CpG
DNA stimulation.  With CFSE labeling to detect division, we usually see
mean time to first division CD27+ B cell precursors at around 72 hours,
and CD27- B cells cells around 98 hours.  Downregulation of IgD occurs
after 1-3 divisions (~48 hours after the first division), downregulation
of CD20 occurs over 6-10 divisions, and upregulation of CD38 over 1-4
divisions.  In addition, you need special conditions for CD138
expression after activation (usually a change in media and cytokines),
which usually occurs after 7-9 divisions at the earliest.

Your experimental design, sampling at 48 hours, is unlikely to show you
any differences.

See our paper:	Huggins, et al. Blood, 15 February 2007, 109:1611-1619. 

Hope this helps,
Martin

_____________________________
Martin S. Zand, MD, PhD
Associate Professor of Medicine, Microbiology and Immunology
Medical Director, Kidney and Pancreas Transplant Programs
Co-Director, Center for Biodefense Immune Modeling
University of Rochester Medical Center
601 Elmwood Ave - Box 675
Rochester, NY 14642
Vox:  585-275-4517
Fax:  585-486-1043
Web:  http://www.urmc.rochester.edu/medicine/nephrology/zandLab.aspx


-----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