[Cytometry] LPS treatment of PBMC

mario picozza mariopicozza at hotmail.com
Fri Apr 12 04:13:06 EDT 2013


Hi Doris, LPS stimulation in PBMC is a strong stimulus, also at very low concentrations, and monocytes are fragile (accounting for cell loss). Also, how do you identify monocytes? In some cases the "classical" MNC gate in fsc/ssc as set in unstim.  is to be extended to high ssc (try to include in the analysis cells on the upper limit of ssc, does the freq. partially restores?, If so, next time reduce the ssc voltage to visualize them) because monocytes very easily gain complexity while differentiating/maturing. In my experience, in PBMC (or whole blood, complex populations in general) stimulations by innate ligands, during 24h there are a lot of changes in the immunophenotype to copy with, and I prefer shorter time points.
Kind Regards

Mario Picozza, Ph.D.
Laboratory of Applied Dermatology
Istituto Dermopatico dell'Immacolata, IDI I.R.C.C.S.
via dei Monti di Creta 104, 00167
Rome, Italy




From: dwiener at health.usf.edu
To: cytometry at lists.purdue.edu
Date: Thu, 11 Apr 2013 13:22:28 +0000
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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