[Cytometry] LPS treatment of PBMC

Gary Warnes g.warnes at qmul.ac.uk
Fri Apr 12 08:56:58 EDT 2013


Hi Doris,	
	Yes Phil is correct, I only stimulated PBMNC and whole blood for 6h
with 10, 100 & 1000 ng/ml of LPS to get a large response, see my paper on
the link
http://www.icms.qmul.ac.uk/flowcytometry/Publications/papers/bjh98.pdf

Hope this helps

Regards

Gary

Dr Gary Warnes
Flow Cytometry Manager 
Flow Cytometry Core Facility
Blizard Institute 
Barts and The London School of Medicine and Dentistry
London University
4 Newark Street 
London
E1 2AT
Tel No.0207-882-2402
Fax No. 0207-882-2180
Website http://www.icms.qmul.ac.uk/flowcytometry/index.html


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Hogarth, Philip
Sent: 12 April 2013 13:22
To: Wiener, Doris; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] LPS treatment of PBMC

Hi,

 

I am no expert, but is 24hrs stim overdoing it & producing lots of cell
death? Are you using a viability stain to look at this?

Perhaps adding β2-ME would help?

 

Phil

 

Phil Hogarth, PhD

Senior Scientist (G7) Immunology

Flow Cytometry Facility Manager

Animal Health and Veterinary Laboratories Agency (AHVLA)

TB Research Department

Woodham Lane, New Haw

Addlestone, KT153NB

 

T: +44 (0) 1932 359557

M: +44 (0) 776 8485613

F: +44 (0) 1932 357260

philip.hogarth at ahvla.gsi.gov.uk

www.defra.gov.uk/ahvla

 

 

 

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Wiener, Doris
Sent: 11 April 2013 14:22
To: 'cytometry at lists.purdue.edu'
Subject: [Cytometry] LPS treatment of PBMC

 

Hi All,

We are treating ficoll isolated PBMC for 24 hours with 100ng/ml LPS and have
noticed the percentage of monocytes in the recovered cells (we detach any
attached monocytes with TryplE) is reduced by 50% or more. We saw  a similar
loss with 10ng/ml LPS. Treatment of parallel cells with PGN does not produce
this effect. Cells are grown in RMPI-1640+glutamine+10% low endotoxin
FBS+pen/strep.  LPS is from Sigma Aldrich 055:B5 (cat#:L6529). Is this seen
by others or do we have a problem? Thanks for your advice as always.

 

Doris Wiener

University of South Florida

Neuropsychiatry Research Lab

Ph:  727-553-3549

Fax: 727-553-3547

 

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