[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

Hope this helps



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 
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



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 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





-----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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