Vegfr 2 pe

To measure circulating EPCs and CECs, a method from Duda et al.18 (link) was adapted with modifications as in our previous study19 (link). Flow cytometry was used for whole blood analysis without enrichment procedures to avoid manipulation artifacts. The EPCs were characterized as CD31⁺ VEGFR-2⁺ CD45^dim CD133⁺; mature CECs were defined as CD31^Bright VEGFR-2⁺ CD45^- CD133^-. The chromophores conjugated with specific antibodies used in this study included CD31-FITC (BD Pharmingen, San Diego, CA), CD133-PE (Miltenyi Biotec, Auburn, CA), CD45-PerCP (BD Pharmingen), and VEGFR2-PE (R&D Systems, Minneapolis, MN). For apoptotic CECs, AAD-7 was also applied to stain the apoptotic CECs. During the analysis of flow cytometry data, the mononuclear cell population was gated to avoid RBC, platelet, cell debris, and neutrophil contamination; 100,000 events in the gated population were collected by a FACScaliber flow cytometer (BD Biosciences, San Jose, CA, USA). The acquisition data were collected and analyzed using CellQuest Software (BD Biosciences).

In order to identify human EPCs, 2 × 10⁵ cells were suspended in 50 μL fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% FBS) then stained with 0.2 mg/mL of each of the following anti-human antibodies: CD31-PE (LifeSpanBioSciences, Seattle, WA, USA), CD34-BV421 (BD Biosciences, San Jose, CA, USA), CD45-BB515 (BD Biosciences), VEGFR-2 PE (R&D Systems, Minneapolis, MN, USA), CD14-FITC (BD Biosciences), and CXCR4-PE (BioLegend, San Diego, CA, USA). OneComp eBeads (Carlsbad, CA, USA) were first stained with 1 μL of each different fluorochrome then used as single-color compensation controls. An unstained sample served as a reference for positive staining. Cells were analyzed using a Cyan flow cytometry (Beckman Coulter, Brea, CA, USA). Single cell data was analyzed using FlowJo, LLC software that gave a percentage for positive cells.

Whole blood (100 µL) was incubated with 5 µL of V500 CD45 (BD Bioscience, San Jose, CA, USA), 20 µL of PerCP-Cy5.5 CD34 (BD Bioscience, San Jose, CA, USA), 5 µL of PE VEGFR-2 (R & D Systems, Minneapolis, MN, USA ), 5 µL APC CD133 (Miltenyi Biotic Inc., Bergisch Gladbach, Germany) and 10 µL of FITC CD144 (BD Bioscience, San Jose, CA, USA) for 30 min. Subsequently, 2 mL of PharmLyse (BD Bioscience, San Jose, CA, USA) were used to lyse the red cells. The sample was then analyzed by flow cytometry on a BD FACS Canto™ II system and the results data processed using BD FACSDiva™ software as described previously [14 (link)].

100 µl of whole blood [6 (link)] was incubated with 5 µl of V500 CD45 (B.D Bioscience), 20 µl of PerCP-Cy5.5 CD34 (BD Bioscience), 5 µl of PE VEGFR-2+ (R&D), 5 µl APC CD133 (Miltenyi Biotec USA), 10 µl of FITC CD144 for 30 min. Subsequently, 2 mls of pharmlyse (BD Bioscience) was used to lyse the red cells. The sample was then analysed by flow cytometry on BD FACS Canto™ II system and results by using BD FACSDiva™ software. On average 450,000 events were counted. cEPCs were defined as CD45^dimCD34⁺VEGFR-2⁺ cells and cECs as CD45^dim, CD133⁻, CD34⁺, and CD144⁺ events. cEPC count was expressed as % leukocytes (Intra-assay variation <8 %) and cECs as per ml of blood.

100 μl of whole blood was incubated with 5 μl of V500 CD45 (BD Biosciences, Oxford, UK), 20 μl of PerCP-Cy5.5 CD34 (BD Biosciences, Oxford, UK), 5 μl of PE VEGFR-2+ (R&D Systems, Roche Diagnostics, West Sussex, UK), 5 μl APC CD133 (BD Biosciences, Oxford, UK), 10 μl FITC CD144 (BD Biosciences, Oxford, UK) for 30 min. Subsequently, 2 ml of pharmlyse (BD Biosciences, Oxford, UK) was used to lyse the red cells. The sample was then analysed by flow cytometry on BD FACS Canto™ II system and samples were run to approximately 500,000 total events. Analysis was carried out using BD FACSDiva™ software. On average 440,000 events were counted. CEPCs events were defined using the most recent definition of CD45^dimCD34⁺ VEGFR2 (KDR)⁺, as recommended by Van Craenenbroeck et al. [21 (link)]. Intra-assay variation (CD45^dimCD34⁺VEGFR-2⁺) was less than 8 %. The results were expressed as % leukocytes [22 (link), 21 (link)]. CECs events were defined as CD45^dimCD133^-CD34⁺CD144⁺ [23 (link), 24 (link)].

The number of cEPCs was assessed by flow cytometry by determining the number of CD34/CD133/VEGFR2-positive cells. In particular, 100 µl of each sample prepared as described in the previous section, were stained with 1 µl each of FITC-CD34 (BD Biosciences, Franklin Lakes, NJ, USA), APC-CD133/2 (Miltenyi Biotec, Bergisch Gladbach, Germany), and PE-VEGFR-2 (R&D Systems, Minneapolis, MN, USA), and incubated in the dark in ice for 15 min. For each sample, a control tube with no antibodies was prepared together with the stained tube. After washing the cells with PBS, FACS analysis was performed on a FacsCanto (BD Biosciences, Franklin Lakes, NJ, USA). The acquisition goal was 2 × 10⁵ events. Samples were analyzed gating a population with morphological characteristics between lymphocytes and monocytes, evaluated on the basis of side scatter and forward scatter parameters. As evident from Figure 1, where representative dot-plots relative to a control and a T1DM patient (belonging to the “<20 y T1DM cohort”) are shown, the cEPC count is reported in Q2, that is a part of the P4 quadrant. Throughout the manuscript, “cEPC counts” refers to the absolute cEPC number per 2 × 10⁵ PBMC.