[Cytometry] LPS treatment of PBMC

Mcfarlin, Brian Brian.Mcfarlin at unt.edu
Fri Apr 12 08:59:37 EDT 2013


It might help if we understood what your end outcome goal is? I would
agree with Phil that the 24-h stimulation seems like a long time unless
your specific measurement application requires that. I think it would be
very wise to use a fixable cell viability dye in your experiment to
determine what type of response you have there.

In terms of your LPS source, we have been using Invivogen as a supplier
for a number of years now. In our experience there LPS preps seem to be a
more pure LPS source. We use them in whole blood cultures as well as
intracellular staining experiments.

On 4/12/13 7:21 AM, "Hogarth, Philip" <Philip.Hogarth at ahvla.gsi.gov.uk>
wrote:

>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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