PMID- 21412982
OWN - NLM
STAT- MEDLINE
DCOM- 20110701
LR  - 20220129
IS  - 1552-4930 (Electronic)
IS  - 1552-4922 (Linking)
VI  - 79
IP  - 4
DP  - 2011 Apr
TI  - A novel flow cytometry-based assay to study leukocyte-endothelial cell 
      interactions in vitro.
PG  - 256-62
LID - 10.1002/cyto.a.21043 [doi]
AB  - Interactions between peripheral blood mononuclear cells (PBMC) and the 
      endothelium critically determine vascular repair and reparative angiogenesis, but 
      also pathological processes, such as atherosclerosis. Current methodology to 
      study these interactions mostly regards PBMC as a homogenous population or it 
      restricts its focus on individual cell types and therefore does not appreciate 
      the differential behavior of individual PBMC subpopulations, which synergize or 
      antagonize each other to obtain the overall effect. We therefore developed a flow 
      cytometry-based in vitro assay to assess multiple parameters of interaction 
      between several individual populations of PBMC and an endothelial monolayer. 
      Freshly isolated, unlabelled human PBMC were left to adhere to or transmigrate 
      through a monolayer of fluorescence-labeled human aortic endothelial cells grown 
      to confluence on the filter membrane of sterile transwell migration inserts. 
      Monocyte chemoattractant protein-1 (MCP-1) was applied as a chemoattractant to 
      the lower compartment of the migration chamber. After 6 h, transmigrating PBMC 
      were harvested from the lower compartment, while nonadherent and adhering cell 
      populations were harvested from the upper compartment by sequential 
      washing/detachment. All three cell fractions were then individually stained with 
      fluorescence-labeled monoclonal antibodies and analyzed by flow cytometry. 
      Quantification was achieved by the usage of counting beads. Endothelial cells 
      were separated from PBMC during the analysis by a multiparametric gating 
      strategy. Using the newly established assay, we observed distinct migration 
      patterns for inflammatory CD14(hi) CD16(neg) and resident CD16(pos) monocytes. 
      These cell types differed in their basal adhesion and transmigration patterns as 
      well as their responses to the CCR2 ligand MCP-1. This assay allows for the 
      parallel study of interactions between multiple individual leukocyte populations 
      and an endothelial layer. Several readouts can be derived from the same 
      experiment, like the composition of adhering and transmigrating cell fractions or 
      the individual adhesion/migration behavior of several distinct cell types.
CI  - Copyright (c) 2011 International Society for Advancement of Cytometry.
FAU - Krankel, Nicolle
AU  - Krankel N
AD  - Department of Cardiology, University Hospital Zurich, Zurich, Switzerland. 
      nicolle.kraenkel@uzh.ch
FAU - Kuschnerus, Kira
AU  - Kuschnerus K
FAU - Madeddu, Paolo
AU  - Madeddu P
FAU - Luscher, Thomas F
AU  - Luscher TF
FAU - Landmesser, Ulf
AU  - Landmesser U
LA  - eng
GR  - G0900912/MRC_/Medical Research Council/United Kingdom
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20110315
PL  - United States
TA  - Cytometry A
JT  - Cytometry. Part A : the journal of the International Society for Analytical 
      Cytology
JID - 101235694
RN  - 0 (Chemokine CCL2)
RN  - 0 (Chemotactic Factors)
SB  - IM
MH  - Cell Adhesion/*physiology
MH  - Cell Communication/*physiology
MH  - Cell Movement
MH  - Cell Separation/methods
MH  - Chemokine CCL2/metabolism
MH  - Chemotactic Factors/metabolism
MH  - Endothelial Cells/cytology/*metabolism
MH  - Endothelium, Vascular/cytology
MH  - Flow Cytometry/instrumentation/*methods
MH  - Humans
MH  - Leukocytes/cytology/*metabolism
EDAT- 2011/03/18 06:00
MHDA- 2011/07/02 06:00
CRDT- 2011/03/18 06:00
PHST- 2010/12/07 00:00 [received]
PHST- 2011/02/01 00:00 [revised]
PHST- 2011/02/13 00:00 [accepted]
PHST- 2011/03/18 06:00 [entrez]
PHST- 2011/03/18 06:00 [pubmed]
PHST- 2011/07/02 06:00 [medline]
AID - 10.1002/cyto.a.21043 [doi]
PST - ppublish
SO  - Cytometry A. 2011 Apr;79(4):256-62. doi: 10.1002/cyto.a.21043. Epub 2011 Mar 15.