Re: Stimulating Human Bcells with CpG

A good paper to guide you in stimulating B cells with  CpG ODN:
Bernasconi NL,	Traggiai E, Lanzavecchia A. 2002. Maintenance of 
Serological Memory by Polyclonal Activation of Human Memory B cells. 
Science 298: 2199.

Also, note that LPS will not work as a positive control for HUMAN B 
cells. Turns out they don't express TLR4.

Cheers,
Lynn Dustin

Gerstein, Rachel wrote:

> 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
>
>
>
>
>
>
>

-- 

Lynn B. Dustin, Ph.D.
The Rockefeller University
Center for the Study of Hepatitis C
Box 64
1230 York Ave. 
New York, NY 10065 USA
Phone: 212-327-7067
Fax: 212-327-7048
http://www.hepccenter.org