Raskit Lachmann and Paola Lanuti contributed equally to first authorship.
OMIP-011: Characterization of circulating endothelial cells (CECs) in peripheral blood
Version of Record online: 30 MAY 2012
Copyright © 2012 International Society for Advancement of Cytometry
Cytometry Part A
Volume 81A, Issue 7, pages 549–551, July 2012
How to Cite
Lachmann, R., Lanuti, P. and Miscia, S. (2012), OMIP-011: Characterization of circulating endothelial cells (CECs) in peripheral blood. Cytometry, 81A: 549–551. doi: 10.1002/cyto.a.22071
- Issue online: 22 JUN 2012
- Version of Record online: 30 MAY 2012
- Manuscript Accepted: 23 APR 2012
- Manuscript Revised: 14 APR 2012
- Manuscript Received: 15 JUN 2011
Additional Supporting Information may be found in the online version of this article.
|CYTO_22071_sm_SuppFig1.tif||6930K||Supplemental Figure 1. Reagent titrations . Reagents were titrated on 4 ml whole peripheral blood, RBC-lysed, from healthy donors. Single FCS files were concatenated to show titrations for each reagent in a single plot. Antibodies and reagents concentrations are displayed along the x-Axis. CD146 and CD106 titrations were carried out by adding CD45 and CD34 antibodies and analyzing CD45-/CD34+ events. Concentrations used in OMIP are highlighted in red.|
|CYTO_22071_sm_SuppFig2.tif||3177K||Supplemental Figure 2. Positive controls. Reagent titrations were performed on other cell types. A CD34-ECD staining (20/100) on cord blood cells. B Staining of CD106 PE-Cy5 (2/100) on TNF-alpha-activated (16 H, 1 ng / ml) HUVEC cells (left plot) compared to control (CTRL – right plot). C Internal controls for CD34 and CD117: CD45+/CD34+/CD117+ cells represent the well-known circulating haemopoietic stem cell compartment. D Internal control for CD146 staining: CD45+/CD146+ cells are peripheral activated-T lymphocytes.|
|CYTO_22071_sm_SuppFig3.tif||11408K||Supplemental Figure 3. Gating strategy and FMO controls. A Cells were not gated on morphological parameters, only artifacts in the upper left and lower right corner were excluded. Aggregates were gated out on a FSC-Area (FSC-A) vs. FSC-High (FSC-H) dot plot. Debris, dead cells, microparticles, and platelets were excluded by gating Syto16+ cells and NiRed- cells. Leucocytes were excluded by gating CD45-, CD45dim and CD31+ cells. CD34+, CD146+, CD106+ and CD117+ cells were separately gated. B Appropriate FMO controls are shown. Dot plots show 8,000 events each, whereas pseudocolour plots and low resolution zebra plots show all analyzed events.|
|CYTO_22071_sm_SuppFig4.tif||4919K||Supplemental Figure 4. Spillover into all detectors. CompBeads were labeled individually with each of 6 fluorochromes and blood samples were stained with Syto16 or NiRed. The percentage of light from the excitation of the primary fluorochrome contaminating each of the other detectors was calculated, as suggested by Perfetto SP et al. (Nat Rev Immunol, 2004). Each bar represents the percentage of the signal subtracted in the given detector, following the compensation procedure.|
|CYTO_22071_sm_SuppTab1.tif||3821K||Supplemental Table 1. Instrument configuration. The panel was optimized for a FACSCanto II with the listed optical elements. Long pass dichroic filters were used throughout the instrument. Light is filtered to each photomultiplier tube such that only wavelengths lower than long pass filter and within the band pass are detected. DPSS – Diode Pumped Solid State, HeNe – Helium-Neon, Cy – cyanine, ECD – energy-coupling dye, LP – long pass, NiRed – LIVE/DEAD fixable near-infrared dead cells stain, PE – R-phycoerythrin.|
|CYTO_22071_sm_SuppTab2.tif||6501K||Supplemental Table 2. Commercially available reagents used in OMIP-011.|
|CYTO_22071_sm_SuppTab3.tif||8048K||Supplemental Table 3. Reagents tested, but not used. FITC – fluorescein isothiocyanate, H7 - Hilite®7, PerCP - peridinin chlorophyl protein-conjugated.|
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