Primary SVF from EAT, SAT, BAT was resuspended in PBS with 2% FBS, and incubated with FACS antibodies for 30 min on ice. Cells were initially selected by size on the basis of forward scatter (FSC) and side scatter (SSC), following separated on the basis of cell-surface markers using a flow cytometer MoFlo XDP (Beckman Coulter, Brea, CA). To detect beige progenitors in SVF, primary SVF was separated on the basis of cell-surface markers including anti-PDGFRα (CD140a)-APC (1:50, BioLegend, San Diego, CA) and anti-CD34-FITC (1:100, eBioscience, San Diego, CA). To quantify MCs in adipose tissues, to isolate MCs from SVF, and to remove MCs from SVF, we stained and sorted MCs with anti-CD34-FITC (1:100, eBioscience), anti-CD45-PE (1:500, eBioscience) and MC markers anti-CD117-APC (1:200, eBioscience) and anti-FCεR1-PE-CY7 (1:200, eBioscience). The MC-removed SVF and MCs (1×105) were collected for TPH1 mRNA expressional analysis.
Anti cd34 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD34-FITC is a fluorescently labeled monoclonal antibody that binds to the CD34 antigen, which is expressed on the surface of hematopoietic stem and progenitor cells. This product is intended for use in flow cytometry applications to identify and enumerate CD34-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 fitc, by Thermo Fisher Scientific (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Anti cd45 percp cy5, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd105 fitc, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (2 mentions)
Annexin 5 binding buffer, by BioLegend (2 mentions)
Annexin 5, by BioLegend (2 mentions)
Cell detachment solution, by Innovative Cell Technologies (2 mentions)
Anti cd90 fitc, by Thermo Fisher Scientific (2 mentions)
Anti cd45 pe, by Thermo Fisher Scientific (1 mentions)
Anti pdgfrα cd140a apc, by BioLegend (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Anti cd117 apc, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Sartorius (1 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Anti cd45 pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti αsma fitc, by Merck Group (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
19 protocols using anti cd34 fitc
1
Isolation and Characterization of Adipose Cell Populations
2
Isolation and Analysis of Murine Mammary and Lung Cells
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Mammary glands and lungs were isolated from mice. Single cell suspensions of mammary glands were prepared as we previously described (Sharon et al., 2013 (link)). For dissociation of lung tissue, collagenase type-II was substituted with trypsin (Biological Industries; 03–051-5B). Cells were stained using the following antibodies: anti-PDGFRα-PE (eBioscience; 12–1401-81), anti-EpCAM-APC (Miltany Biotec; 130–102-234), anti-CD45-PerCP-Cy5.5 (eBioscience; 45–0451-82), anti-CD45-PE-Cy7 (eBioscience; 25–0451-82), anti-Annexin V-Alexafluor 488 (Invitrogen; A13201 ), anti-CD34–FITC (eBioscience; 11–0341-82), and anti-αSMA-FITC (Sigma; F3777). For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm Plus kit according to the manufacturer's protocol. DAPI was used to exclude dead cells (Molecular Probes; D3571). Analysis or sorting was done with BD FACSAria II, BD FACSAria Fusion or CytoFLEX Flow Cytometer (Beckman Coulter, Inc.). Data analysis was done with BD FACSDiva software (BD Biosciences), CytExpert 2.0 (Beckman Coulter, Inc.) or the Kaluza Flow Analysis software (Beckman Coulter, Inc.).
3
Immunophenotyping of MSCs and Endothelial Cells
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We used trypsin (0.05%) and EDTA (0.02%) to remove the MSCs and differentiated endothelial cells from the culture flasks. The cells were counted with a Neubauer slide and cell viability determined with trypan blue staining. The cell suspension (106 cells/ml) was prepared in 50 μl PBS and incubated with either fluorescein isothiocyanate (FITC) or PE-conjugated antibodies, in the dark for 45 minutes at 4˚C. Immud nophenotyping of the MSCs was carried out using anti-CD44-PE, anti-CD166-PE, anti-CD105-FITC, and anti-CD34-FITC antibodies (eBioscience, USA). In order to immunophenotype the endothelial cells before and after differentiation, we incubated the MSCs and differentiated endothelial cells with anti-FLT1FITC, anti-VE-cadherin-FITC, anti-VCAM1-FITC, anti-Tie2-FITC, anti-vWF-FITC, anti-CD31-FITC, and anti-VEGFR2-FITC antibodies (eBioscience, USA). Next, the cells were washed twice with PBS that contained 2% bovine serum albumin (BSA, Gibco Invitrogen, USA) and fixed with 1% paraformaldehyde solution in PBS. Mouse isotype antibodies served as the negative control. Analysis was performed using a flow cytometer (Partech, Germany).
4
Flow Cytometry of Adipose-Derived Stem Cells
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Cell phenotype detection was performed as previously reported [38 (link)]. After reaching 80–90% confluence, P3 ADSCs were digested with 0.25% pancreatin-0.02% ethylenediamine tetraacetic acid (EDTA; T1300, Solarbio), and then no less than 2 × 105 cells were collected from each sample for analysis. Next, cells were resuspended in 100 μL of PBS and subjected to centrifugation at 1,000 r/min for 5 min. After removing the PBS, cells were resuspended in 200 μL of PBS, incubated for 30 min with antibodies, and washed twice with PBS. After removing the PBS again and resuspending the cells with 300 μL PBS, the cell suspension was analyzed by flow cytometry (Accuri™ C6, BD Biosciences, USA). The following monoclonal antibodies used in this experiment were purchased from eBioscience™ of Thermo Fisher Scientific: anti-CD29-FITC (#11-0291-80), anti-CD90-FITC (#11-0909-42), anti-CD73-FITC (#11-0739-41), anti-CD105-FITC (#MA1-19594), anti-CD34-FITC (#11-0349-41), and anti-CD45-FITC (#11-9459-41) antibodies.
5
Multicolor Flow Cytometry of Hematopoietic Cells
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Cells were isolated from BM by flushing marrow cavities. Cells were isolated from FL from timed pregnancies. Cells were analyzed on a Fortessa flow cytometer or sorted on a FACS Aria (BD). The following anti–mouse fluorescence-conjugated antibodies were used: anti-B220, CD3, Ter119, Mac-1, Gr-1 conjugated to Pacific blue as a lineage cocktail, anti–c-kit–APC-Cy7, anti-CD34–FITC, and anti-CD16/32–PerCp-Cy5.5 (eBioscience and Affymetrix); anti–Sca-1–PE-Cy7; anti-Ter119–APC and anti–Gr-1–PE; anti–Mac-1–PE-Cy5 and anti-CD150–APC (BioLegend); and anti–Mac-1–AF700, anti-CD48–PE, and anti-CD71–FITC (BD). Cells were counterstained for viability with Sytox blue or propidium iodide (Thermo Fisher Scientific). Viability for cell quantification was determined using a parallel tube stained with Sytox blue.
6
Multimodal Embryonic Tissue Analysis
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Cryosections were prepared from paraformaldehyde-fixed embryos and stained with anti-Mll1 (Novus Biologicals and Bethyl Laboratories), anti-CD34-FITC (eBioscience), anti-ckit (R&D Systems), followed by secondary antibodies anti-rabbit AlexaFluor555, anti-goat-AlexaFluor555, and anti-rabbit AlexaFluor647 (Abcam). Sections were mounted with DAPI-containing Vectashield mounting medium (Vector Laboratories), images were obtained on an AxioImager Z2 (Zeiss) fitted with a Hammamatsu Flash 4 V2 camera at room temperature, and images were analyzed with the Zen Blue 2012 software.
Tissues from diseased mice were fixed in formalin and embedded in wax. Sections were prepared and stained with H&E, dehydrated, and mounted in DePeX-mounting medium (Fisher Scientific).
Tissues from diseased mice were fixed in formalin and embedded in wax. Sections were prepared and stained with H&E, dehydrated, and mounted in DePeX-mounting medium (Fisher Scientific).
7
Cytokine and Fatty Acid-Induced Apoptosis in Mouse Hair Follicle Stem Cells
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mHFSC were grown to confluence in a 24-well plate at least 12 h before experiments. For cytokine-induced apoptosis assay, mHFSCs were added with IL-17, IFNγ, TNFα at a range of physio- pathological levels (from 0 to 200 ng/mL) into the HFSC culture Complete Growth Medium for 24 h. To monitor FA-induced apoptosis, mHFSCs were added with 200 μM DPA, EPA, or control BSA into the medium for 24 h. Cells were gently harvested using Cell Detachment Solution (Accutase, AT-104, Innovative Cell Technologies), washed with Annexin V Binding Buffer (422201, Biolegend) twice and stained with Annexin-V (640941 for cytokine-induced apoptosis and 640947 for FA-induced apoptosis from Biolegend, respectively) in Binding Buffer for 20 min. For in vivo HFSC staining, dermal cells isolated from mice fed the LFD, fish oil HFD, or cocoa butter HFD were stained with anti-CD34-FITC (11-0341-082, eBioscience) and Zombie blue. All samples acquired using FACS LSR Fortessa flow cytometer. Data were analyzed using FlowJo software.
8
Comprehensive Flow Cytometry Analysis of Mouse Hematopoietic Cells
9
Isolation and Characterization of Human MSC
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The bone marrow samples were obtained from the Second Affiliated Hospital of Nanchang University, China. Informed consent was obtained from all patients included in the study. MSC were isolated and cultured in DMEM/F12 (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 2.0 mM glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) at 37°C in a humidified atmosphere containing 5% CO2.
The immunophenotype of MSC was analyzed by cytofluorimetric analysis. Before experiments, the following monoclonal antibodies were used: anti-CD105-PE, anti-CD34-FITC, anti- CD45-FITC, and anti-CD90-PE (eBioscience, USA). BD FACSDiva flow cytometry (BD FACS Canto™ II, USA) was used. The adipogenic, osteogenic, and chondrogenic differentiation abilities of MSC were determined as previously described.
The immunophenotype of MSC was analyzed by cytofluorimetric analysis. Before experiments, the following monoclonal antibodies were used: anti-CD105-PE, anti-CD34-FITC, anti- CD45-FITC, and anti-CD90-PE (eBioscience, USA). BD FACSDiva flow cytometry (BD FACS Canto™ II, USA) was used. The adipogenic, osteogenic, and chondrogenic differentiation abilities of MSC were determined as previously described.
10
Isolation of CD34+/VEGFR2+ Cells by FACS
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Cell sorting was performed on a FACSAria II (BD Biosciences, San Jose, CA) using a 488 nm laser line (chosen for its compatibility with fluorescein isothiocyanate [FITC]) for light scattering and fluorescence excitation. Standard emission filters for FITC and R- phycoerythrin filter were used. To isolate the Cluster of differentiation molecule - 34 and vascular endothelial growth factor receptor -2 positive cells (CD34+/VEGFR2+) cells from other mononuclear precursor cells, the sample was blocked with 10% goat serum (Gibco, Carlsbad, CA) and labeled with the following fluorescent antibodies for 20 min each at 4 °C: anti-VEGFR-2-phycoerythrin (1:200, BD PharMingen, San Jose, CA) and anti-CD34-FITC (1:200, eBioscience, San Diego, CA). Cells were then sorted with flow cytometry (BD Biosciences FACSAria, University of Chicago Flow Cytometry Facility, Chicago, IL) to isolate double positive cells exhibiting both cell surface markers.
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