[Cytometry] LPS treatment of PBMC

Denis Polancec Denis.Polancec at glpg.com
Fri Apr 12 08:39:38 EDT 2013


Hi, 
I agree with Phil.
You are using a quite high LPS concentrations.
Try to titrate LPS using 5-fold dilutions starting from 10 ng/ml.
Always in staining use viabilty dye. 
Best wishes,

Denis

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Hogarth, Philip
Sent: Friday, April 12, 2013 2:22 PM
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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