[Cytometry] LPS treatment of PBMC

Robin Barclay robin.barclay at ed.ac.uk
Fri Apr 12 09:23:53 EDT 2013


LPS requires LBP (from plasma/serum) to interact via the CD14
LPS-receptor, and in conjunction with LBP it is active at very low
(picogram/femtogram) levels. However plasma/serum contains LPS
neutralising antibodies which can mask some of the LPS activity, so
maybe you need higher (nanogram range) amounts in the presence of serum.
The activation will be rapid so extended incubation is pointless (in
vivo you see plasma cytokine responses typically at 6h following LPS
exposure), but you don't state what outcome you are looking at. If LPS
doesn't interact in conjunction with LBP it just loads into the cell
membrane through its hydrophobic lipid A but I don't know what that
triggers, if anything. Are you counting total cells or only adherent
cells (i.e. do you decant/wash then recover adherent) - many monocytes
may be becoming non-adherent but not dying and could be lost if you are
decanting.
The purity of the LPS doesn't matter - in fact some bacterial OMP might
be beneficial.
G Robin Barclay
Edinburgh

On 12/04/2013 13:59, Mcfarlin, Brian wrote:
> 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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