Quantification of peripheral circulating eEPC and analysis of eEPC regeneration was performed as described in numerous previous studies [
11,
13,
18,
19,
22]. Nevertheless, in order to supply a better understanding of the parameters used for detecting eEPCs from the blood and for evaluating eEPC regeneration, the methodical approaches shall be outlined more in detail. Quantification of peripheral circulating eEPCs was performed by flow cytometry. Mononuclear cells (MNCs) were isolated by density gradient centrifugation using Histopaque-1077 solution (Sigma Diagnostics, St. Louis, MO) from ≈ 7.5 ml of heparinized peripheral blood. Cells were initially incubated for 1 h (on ice) using the following antibodies: rabbit anti CD133 (ab16518 – Abcam, Cambridge, UK), mouse anti human VEGFR2 (Vacular Endothelial Growth Factor Receptor 2 - KDR, directly conjugated - FAB 3571 F – R&D Systems, Minneapolis, MN, USA), followed by secondary incubation with PE-conjugated goat anti rabbit Fab (VEGFR, 111-116-144 – Jackson Immunoresearch, Baltimore, PA, USA) for 30 min on ice, respectively. After incubation, cells were washed with PBS-BSA 1 % (w/v). Data were acquired using a FACScalibur cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a 488 nm argon laser and a 635 nm red diode laser and analyzed using CellQuest software (Becton Dickinson, San Jose, CA, USA). The setup of FACScalibur was performed according to the manufacturer’s instructions using unstained and single-antibody stained cells. Specificity of staining was controlled by incubation with isotype-matched immunoglobulins. To quantify eEPCs, the numbers of CD133/KDR double-positive cells within the myelomonocytic cell population were counted. eEPC regeneration on the other hand was evaluated by a Colony-Forming Units (CFU) assay. The assay was performed by using the EndoCult Liquid Medium Kit® (StemCell Technologies, Vancouver, BC, Canada) per the manufacturer’s protocol. MNCs were resuspended in complete EndoCult medium and seeded at 5 × 10
6 cells/well on fibronectin-coated tissue culture plates (BD Biosciences, Rockville, MD, USA). After 48 h, wells were washed with media and nonadherent cells were collected. Nonadherent cells were plated in their existing media at 10
6 cells/well in 24 well fibronectin-coated tissue culture plates for three days. Only colonies with at least 20 cells, containing rounded cells in the middle and elongated cells at the periphery, were considered as CFU-EC (Colony Forming Unit-Endothelial Cell) colonies. The numbers of colonies (colonies/well) appearing after this period were counted. At least two members of the laboratory staff evaluated the numbers. They were blinded for the diagnosis and status of the investigated patients/controls. The phenotype of cells within the colonies was determined more in detail. For this purpose, cells were characterized by the uptake of DiI-labeled acetylated low density lipoprotein (Dil-acLDL) (Invitrogen, Carlsbad, CA, USA) and binding of FITC-labeled UE lectin (Sigma Diagnostics, St. Louis, MO). Cells were first incubated with 10 μg/ml DiI-ac-LDL at 37 °C for 1 h and later fixed with 2 % formaldehyde for 10 min, followed by incubation with UE(Ulex Europaeus) lectin at 37 °C for 1 h. In some experiments, cells were additionally stained for FSP-1 (see below). The number and of Dil-acLDL+/UE lectin +/ FSP-1+cells was counted by laser scanning microscopy using an inverted fluorescence microscope IX-71 (Olympus Deutschland GmbH, Hamburg, Germany) equipped with the appropriate excitation and emission filters (AHF Analysentechnik, Tübingen, Germany). In order to provide some information about concistency of the CFU-EC assay, we compared the cumulative mean colony numbers aquired from healthy controls evaluated in three different studies from the past [
19,
22]. One of the three studies has not been published yet. The mean number of colonies in all studies was 29.9 ± 2.2. It did not significantly differ from the current mean in healthy controls (
p = 0.15).