Tumor specimens were prepared as single cell suspensions as previously described [48 ]. Cells from tumor specimens were immunostained with CD45-APC-Cy7, CD31-APC, and VEGFR2-PE (BioLegend, San Diego, CA). Endothelial cells were defined as CD45–/CD31+/VEGFR2+. At least 100,000 events were collected for each sample. The experiments were performed using Cyan-ADP flow cytometer and analyzed by Summit Version 4.3 software (Beckman Coulter, Switzerland).
Vegfr2 pe
Manufactured by BioLegend
VEGFR2 PE is a fluorochrome-conjugated antibody that binds to the vascular endothelial growth factor receptor 2 (VEGFR2) protein. VEGFR2 is a cell surface receptor that plays a crucial role in the regulation of angiogenesis, the process of new blood vessel formation.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (2 mentions)
Cd31 fitc, by BioLegend (2 mentions)
Cd90 fitc, by BioLegend (2 mentions)
Cd11b apc, by BioLegend (2 mentions)
Cd146 pe, by BioLegend (2 mentions)
Cd73 apc, by BioLegend (2 mentions)
Cd45 bv605, by BioLegend (2 mentions)
Cd34 apc cy7, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Cd31 apc, by BioLegend (1 mentions)
Summit version 4, by Beckman Coulter (1 mentions)
Cyan adp flow cytometer, by Beckman Coulter (1 mentions)
Collagenase, by Worthington (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vegfr2, by BioLegend (1 mentions)
3 protocols using vegfr2 pe
1
Enumeration of Tumor Endothelial Cells
2
Phenotypical Analysis of Stromal Vascular Fraction
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The SVF cell phenotype was determined by 6-channel cytofluorimetric analysis before and after the culture. For this latter condition, constructs were digested in 0.15% w/v collagenase (Worthington Biochemical Corporation) and cells were collected by centrifugation. Cells in suspension were then incubated for 30 min on ice with different fluorochrome-conjugated antibodies to human CD90 FITC, CD73 APC, CD31 FITC, CD34 APC-Cy7, CD146 PE, CD45 BV605, VEGFR2 PE, and CD11b APC (all from Biolegend) in staining buffer (PBS, 0.5% v/v FBS, 2 mM EDTA). 5 µl per million cells were used for all the antibodies according to the manufacturer’s protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the live cells (DAPI positive) or ratio over the fresh SVF population.
3
Comprehensive Characterization of Freshly Isolated SVF Cells
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The characterization of freshly isolated SVF cells from three donors was performed by using 6-channel cytofluorimetric analyses. Cell suspension was incubated for 30 min on ice with different fluorochrome-conjugated antibodies to human CD90 FITC, CD73 APC, CD31 FITC, CD34 APC-Cy7, CD146 PE, CD45 BV605, VEGFR2 PE, and CD11b APC (all from Biolegend) in staining buffer (PBS, 0.5% v/v FBS, 2mM EDTA).
Stromal mesenchymal cells, progenitor and mature endothelial cells, and myeloid cells were identified as CD45 -CD73 + CD90 + , CD45 -CD31 + CD34 + , CD45 -CD31 + VEGFR2 + , and CD45 + CD11b + , respectively. Pericytes were identified as CD45 -CD34 - CD146 + , as previously described [31] . 5 µl per million cells were used for all the antibodies according to the manufacturer's protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the living cells, positive stained for (4',6-diamidino-2-phenylindole) DAPI (Invitrogen).
Stromal mesenchymal cells, progenitor and mature endothelial cells, and myeloid cells were identified as CD45 -CD73 + CD90 + , CD45 -CD31 + CD34 + , CD45 -CD31 + VEGFR2 + , and CD45 + CD11b + , respectively. Pericytes were identified as CD45 -CD34 - CD146 + , as previously described [31] . 5 µl per million cells were used for all the antibodies according to the manufacturer's protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the living cells, positive stained for (4',6-diamidino-2-phenylindole) DAPI (Invitrogen).
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