For flow cytometry analysis, cells were washed in PBS and subjected to immunofluorescence (IF) staining with anti-CD34-PE-Cy7 (BD Pharmingen, #556626), a rat monoclonal anti-human DLK1/Pref1 (AdipoGen, #AG-25A-0091) along with anti-rat-APC (BD Pharmingen, #5510109), anti-CD31-FITC (BD Pharmingen, #555445), anti-CD45-APC-Cy7 (BD Pharmingen, #561863) and anti-DPP4/CD26 (Biolegend, #302715). Cells were measured using a FACS Canto II (BD Bioscience), and data were analysed using FlowJo 10.5.2 software. For ex vivo analysis of ASC, flow cytometric sorting of freshly isolated SVFs was performed. Therefore, cells were subjected to IF staining with following antibodies: anti-CD34-PE-Cy7, a rat monoclonal anti-human DLK1/Pref1 along with anti-rat-APC, and anti-DPP4/CD26, and sorted using a FACS Aria (BD Bioscience). For cell count experiments, events recorded during 90 s were assessed, and separate biological triplicates were measured.
Anti cd34 pe cy7
Manufactured by BD
Sourced in United States
Anti-CD34-PE-Cy7 is a fluorescently conjugated monoclonal antibody that binds to the CD34 antigen. CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. This product can be used for the identification and enumeration of CD34-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (3 mentions)
Anti cd90 pe, by Thermo Fisher Scientific (3 mentions)
Anti lin fitc, by Thermo Fisher Scientific (3 mentions)
Anti cd45ra apc h7, by BD (3 mentions)
Anti cd45 fitc, by Beckman Coulter (2 mentions)
Anti cd34 pe, by Beckman Coulter (2 mentions)
Anti cd38 apc, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (1 mentions)
Anti cd45 apc cy7, by BD (1 mentions)
Anti cd31 fitc, by BD (1 mentions)
Facsaria cell sorting system, by BD (1 mentions)
Lymphoprep, by Fresenius (1 mentions)
Stemcare cb collect blood bag system, by Fresenius (1 mentions)
Cd45 apc cy7, by BD (1 mentions)
Diva software, by BD (1 mentions)
Anti cd73 pe, by BD (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
10 protocols using anti cd34 pe cy7
1
Flow Cytometric Characterization of ASC
2
Isolation of Hematopoietic Stem Cells
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Umbilical cord blood was collected in several hospitals using Stemcare/CB collect blood bag system (Fresenius Kabi Norge AS) containing citrate phosphate dextrose (CPD) as an anticoagulant. Approval for collection was obtained from the Medical Ethical Committee of the Erasmus University Medical Centre (MEC-2009–410) and written informed consent from the mother was obtained prior to donation of the cord blood. Within 48 hours after collection, mononuclear cells were isolated using ficoll (Lymphoprep, Fresenius Kabi Norge AS). CD34+ cells were isolated with double positive immunomagnetic selection using Magnetic Activated Cell Sorting (MACS) technology according instructions of the manufacturer (Miltenyi Biotech GmBH, Bergisch Gladbach, Germany). MACS-selected CD34+ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI-Lin-CD34+CD38lowCD45RAlowCD90+-cells, highly enriched for hematopoietic stem cells (HSC)[30 (link)], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA).
3
Multimarker Characterization of Mesenchymal Stem Cells
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After the isolation, characterization of MSCs was completed using anti-CD34 PE-Cy7 (BD Biosciences, San Diego, CA, USA), CD45 APC-Cy7 (BD Biosciences Pharmingen, San Diego, CA, USA), anti-CD73 PE (BD Biosciences Pharmingen), and anti-NG2 PE (Beckman Coulter, Marsellia, France) antibodies by flowcytometry (BD FACSCanto-II, USA) and DiVA software (BD Biosciences Pharmingen) (10 ,11 (link)).
4
Quantifying Cell Labeling Efficiency and Intensity
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Labeling efficiency and median labeling intensity were determined using flow cytometry. Cells were stained with anti-CD45-APC-H7, anti-CD34-PE-Cy7 (both from BD Biosciences, San Jose, CA, USA) and diamidinophenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA). Only viable (DAPI-) CD45+CD34+ cells were included in the analysis. The maximal fluorescence intensity of mock-labeled control cells in the FITC-channel was set as threshold for considering a cell labeled. Flow cytometric analysis was performed using a BD FACSCanto™ (BD Biosciences, San Jose, CA, USA) and data was analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).
To assess 19F-PLGA labeling intensity in cells that divided 0–3 times, cells were labeled with 5μM CellTrace Violet (Molecular Probes, Eugene, Oregon, USA) upon 19F-PLGA labeling and subsequently cultured for 4 days in our culture medium as described above.
To assess 19F-PLGA labeling intensity in cells that divided 0–3 times, cells were labeled with 5μM CellTrace Violet (Molecular Probes, Eugene, Oregon, USA) upon 19F-PLGA labeling and subsequently cultured for 4 days in our culture medium as described above.
5
FACS Analysis of hESC-Derived Stem Cells
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hMSCs derived from untransduced and transduced hESCs were resuspended in FACS buffer and stained with anti-CD105-APC antibodies (eBioscience, San Diego, CA) and analyzed by FACS. eGFP expression was measured in the CD105+ population. Similarly, hematopoietic cells derived from untransduced and transduced hESCs were resuspended in FACS buffer and stained with anti-CD34-PE-Cy7 (BD Biosciences) and anti-CD45-APC (Miltenyi Biotech, Bergish Gladbach, Germany) antibodies. Live cells identified by 7-AAD viability dye exclusion were analyzed for surface-marker and eGFP expression using a FACS Canto II. The populations analyzed were hemogenic progenitors (CD45− CD34+), hematopoietic progenitors (CD45+CD34+), and mature blood cells (CD45+CD34−).
6
Quantification of Human Hematopoietic Stem Cells
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Absolute numbers of viable human CD34+ cells were determined by a single platform flow cytometric assay using anti‐FITC‐CD45, anti‐CD34‐PE, and Stem‐Count Fluorospheres from the Stem‐Kit Reagents kit (Beckman Coulter) and DAPI (Sigma). The frequencies of human phenotypic HSCs were determined using anti‐Lin‐FITC, anti‐CD38‐APC, anti‐CD90‐PE (Thermo Fisher Scientific), anti‐CD34‐PE‐Cy7, anti‐CD45RA‐APC‐H7 (BD Biosciences), and DAPI. All samples were analyzed using BD FACSCanto II (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Inc, Ashland, OR, USA).
7
Quantifying Human Hematopoietic Stem Cells
8
Immunophenotyping of Hematopoietic Cells
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Annexin V-V450, anti-CD138-APC, anti-CD41a-PE, anti-P-gp-FITC, anti-CD34-PE-Cy7, matched isotype controls, BDTM CompBeads anti-mouse-Ig k, SpheroTM Rainbow calibration particles and TruCountTM tubes were from BD Biosciences (Sydney, NSW, Australia). Latex beads of 0.3 and 1.1 µm diameter were from Sigma-Aldrich (Sydney, NSW, Australia). Details of other reagents used are described in Supplementary Table 1 .
9
Isolation and Identification of SVF Cells
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The SVF was isolated as described above and cells were directly subjected to immunofluorescence staining with anti-CD34-PE-Cy7 (BD Pharmingen, #556626), a rat monoclonal anti-human-DLK1/PREF1 antibody (Adipogen, AG-25A-0091) along with an anti-rat-APC antibody (BD Pharmingen, #551019) and anti-CD24-BV421 (Biolegend, #311121). Cells were sorted using a FACS Aria machine (BD, Bioscience, Vienna, Austria) and collected in ASC2 medium. Directly after sorting, cells were counted and immediately used for experiments.
10
Quantifying Endothelial Cells in Bone Marrow
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BM mononuclear cells (BMMNCs) were isolated with Ficoll-Paque solution (HaoYang, Tianjin, China). The following multicolour flow cytometry panel were applied to quantify ECs in BM: anti-CD34-PE/Cy7, anti-CD309-PE, and anti-CD133-APC (BD Biosciences, CA, USA). ECs were identified as CD34 + CD309 + CD133 + , and the relative EC frequency was expressed as a fraction of BMMNCs [23] [24] [25] [26] [27] . Samples were collected on a BD LSRFortessa flow cytometer and the compensation adjustion as well as data analysis were accomplished via a BD LSRFortessa software.
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