WJ-MSCs and HUVECs, respectively, at the eighth and the sixth passage, were treated with 0.05% trypsin–EDTA and collected; 106 cells per sample were incubated with 1 μg of the specific antibody, conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), phycoerythrin-cyanine 5.5 (PE Cy5.5), or Alexa Fluor 488 for 30 min at 4°C in the dark. WJ-MSCs were stained using the following antibodies: anti-CD31, anti-CD73, anti-CD13, anti-CD90, anti-CD117, anti-CD14, anti-CD34, anti-CD105, anti-CD146, anti-CD133, anti-CD144, anti-ESA, anti-HLA-ABC, anti-HLA-DR, anti-CD45 (Becton Dickinson [BD], San Jose, CA), anti-CD29, anti-CD44, and anti-CD166 (Ancell, Bayport, MN). HUVECs were stained with anti-CD146 (BD) and anti-CD144 (Acris Antibodies, San Diego, CA). After incubation, cells were washed and acquired with a flow cytometer (FACS Calibur; BD), collecting 10,000 events per sample. Data were analyzed by the FlowJo software v8.8.6 (TreeStar, Ashland, OR). The mean fluorescence intensity (MFI) ratio values were calculated (i.e., dividing the MFI of positive events by the MFI of negative events).22 (link),23 (link)
Anti cd117
Manufactured by BD
Sourced in United States
Anti-CD117 is a laboratory reagent used in flow cytometry applications. It is a monoclonal antibody that specifically binds to the CD117 surface antigen, also known as c-Kit. The core function of Anti-CD117 is to detect and identify cells expressing the CD117 receptor.
Automatically generated - may contain errors
Lab products found in correlation
Anti hla dr, by BD (5 mentions)
Anti cd90, by BD (4 mentions)
Anti cd34, by BD (4 mentions)
Anti cd45, by BD (4 mentions)
Anti cd146, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Anti fcεri, by BD (3 mentions)
Anti tlr2 monoclonal antibodies, by BD (3 mentions)
Collagenase type 4, by Thermo Fisher Scientific (3 mentions)
Collagenase type 2, by Worthington (3 mentions)
Anti cd73, by BD (2 mentions)
Anti cd144, by OriGene (2 mentions)
Anti cd31, by BD (2 mentions)
Anti cd14, by BD (2 mentions)
Anti cd44, by BD (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Anti cd105, by BD (1 mentions)
Anti cd13, by BD (1 mentions)
Anti oct3 4, by BD (1 mentions)
Anti cd34, by Beckman Coulter (1 mentions)
13 protocols using anti cd117
1
Flow Cytometric Characterization of MSCs and HUVECs
2
Characterization of hPDLSCs by Flow Cytometry
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hPDLSCs at the second passage were collected; 5 × 105 cells per sample were incubated with 1 μg of the specific antibody, conjugated with fluorescein isothiocyanate, phycoerythrin, allophycocyanin, phycoerythrin-cyanine 5.5, or Alexa Fluor 488 for 30 min at 4 °C in the dark. hPDLSCs were stained using the following antibodies: anti-CD13, anti-CD29, anti-CD44, anti-CD45, anti-CD105, anti-CD166 (Ancell, MN, USA), anti-CD14, anti-CD133 (BergischGladbach, Germany), anti-CD73, anti-CD90, anti-CD117, anti-CD146, anti-CD271, anti-Sox2, anti-HLA-DR, anti-SSEA4, anti-OCT3/4 (Becton Dickinson, BD, San Jose, CA, USA), anti-CD144 (Acris Antibodies, Herford, Germany) and anti-CD34 (Beckman Coulter, Fullerton, CA, USA). After incubation, cells were acquired with a flow cytometer (FACS Calibur; BD). Data were analyzed by the FlowJo software v8.8.6 (TreeStar, Ashland, OR, USA) [17 (link)].
3
Culture and Purification of Mast Cells
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LAD-2 cells were cultured as described (Kirshenbaum et al., 2003 (link); Radinger et al., 2010 ) in StemPro-34 medium containing 13 ml of StemPro-34 Nutrient Supplement and l -glutamine (2 mM) and penicillin (100 U/ml)/streptomycin (100 μg/ml; all from GIBCO, Grand Island, NY) with 100 ng/ml recombinant human SCF added (Peprotech, Rocky Hill, NJ). Half of the medium supplemented with SCF was changed every 7 d. HLMCs were obtained by lung resection for bronchial carcinoma and purified using immunoaffinity magnetic selection using anti-CD117 (BD Biosciences, San Jose, CA) as described (Sanmugalingam et al., 2000 (link)) and were cultured as described (Cruse et al., 2008 (link)). All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee, United Kingdom. Human peripheral blood–derived mast cells were cultured from CD34+ progenitors as described (Radinger et al., 2010 ). The donors provided an informed consent, and cells were obtained under a protocol (NCT00001756) approved by the National Institute of Allergy and Infectious Diseases, National Institutes of Health Internal Review Board.
4
Multimarker Flow Cytometry Analysis
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For flow cytometry analysis, 2 × 105 cells were washed with phosphate-buffered saline (PBS) and then stained with the following antibodies: anti-CD11b (BD Pharmingen, Franklin Lakes, NJ, USA), anti-CD29 (eBioscience, San Diego, CA, USA), anti-CD31 (BD Pharmingen), anti-CD34 (BD Pharmingen), anti-CD44 (BD Pharmingen), anti-CD45 (BD Pharmingen), anti-CD73 (eBioscience), anti-CD105 (eBioscience), anti-CD90 (BD Pharmingen), anti-CD117 (BD Pharmingen), anti-Sca1 (BD Pharmingen), anti-CD26 (eBioscience), anti-EpCAM (Abcam), and goat anti-rabbit IgG PE-Cy5.5 (Thermo Fisher Scientific).
5
Multiparametric Flow Cytometry Analysis
6
Multiparametric Flow Cytometry for Cell Sorting
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Peridinin chlorphyll protein (PerCP) anti CD45 (pan-leukocyte antigen), phycoerythrin-conjugated antibodies anti-CD117 (for precursors), fluorescein isothiocyanate- conjugated (FITC) anti-CD15 and anti-CD33 (for myeloid cells), Texas Red conjugated anti-CD3 (for T-lymphocytes) and allophycocyanin-cyanin7-conjugated (APC-Cy7) anti-CD19 (for B-lymphocytes) were used for sorting (BD Biosciences, Milan, Italy). Staining of healthy donor BM cells was performed in standard conditions, and sorting was performed using FACSAria (BD Biosciences). Data were analyzed using FACS Diva Software (v8.0.1, BD Biosciences).
7
Multiparameter Flow Cytometry Analysis
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Phenotypic analyses were performed in FACSCalibur and FACSCanto II (BD Biosciences) flow cytometers using the following mAbs: PE-labeled anti-CD1a, anti-CD34, anti-CD127, and anti-TCRαβ (Beckman Coulter); anti-CD3, anti-CD13, anti-CD44, anti-CD45RA, anti-CD123, anti-CD135, anti-HLA-DR, and anti-Notch4 (BD Biosciences); anti-CD116 (Immunotech); anti-Notch1 and anti-Notch3 (BioLegend); PC5-labeled anti-CD7 (Caltag); anti-CD4, anti-CD5, anti-CD13, anti-CD33, and anti-CD34 (Beckman Coulter); BB515-labeled anti-CD115 (BD Biosciences); FITC-labeled anti-BDCA1 and anti-BDCA2 (Miltenyi Biotec); anti-CD11c (Life Technologies) and anti-CD5, anti-CD7, anti-CD8, anti-CD14, and anti-CD44 (BD Biosciences); V450-labeled anti-CD45; and APC-labeled anti-CD34 (Beckman Coulter) and anti-CD117 (BD Biosciences). Irrelevant isotype-matched Igs (Caltag) were used as controls. Staining with biotin-coupled Annexin V (Roche) plus streptavidin-PE (Invitrogen) and 7-AAD (BD Biosciences) was used for apoptosis analysis. Cell proliferation was quantified by flow cytometry using CFSE labeling, following the manufacturer’s instructions (BD Biosciences).
8
Isolation and Flow Cytometry of Skin Cells
9
Isolation and Analysis of Skin Immune Cells
10
Anticancer Properties of WIN-55 Explored
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Cell lines were cultured (5 × 105 cells/well) at the indicated concentrations of WIN-55 or DMSO in triplicate wells for 18, 48 and 72 h. Isolated primary cells were cultured (5 × 105 cells/well) for 18 h with DMSO (control) or WIN-55 with or without CB antagonists. WIN-55 was added at different concentrations and time-points. Cell viability was determined by Cell Counting Kit (CCK-8) assay as per manufacturer’s instructions (Dojindo Molecular Technologies). Optical densities were measured at 450 nm using a plate reader MultiskanTM Go Microplate (Thermo Fisher Scientific, Waltham, MA, USA).
AML cells from patients’ BM were identified at diagnosis with a combination of monoclonal antibodies against AML cells-associated antigens at diagnosis (anti-CD33, anti-CD34, anti-CD117, and anti-CD45 [BD Biosciences]). Apoptosis was assessed by Annexin V/7AAD staining assay kit, as per manufacturer’s instructions (BD Biosciences) and analysed on a FACS Canto II Flow Cytometer (BD Biosciences) and analysed with InfinicytTM Software (Cytognos, Spain).
Other studies, such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), western blot, mitochondrial damage or enzyme activity assays are detailed in the supplementary files.
AML cells from patients’ BM were identified at diagnosis with a combination of monoclonal antibodies against AML cells-associated antigens at diagnosis (anti-CD33, anti-CD34, anti-CD117, and anti-CD45 [BD Biosciences]). Apoptosis was assessed by Annexin V/7AAD staining assay kit, as per manufacturer’s instructions (BD Biosciences) and analysed on a FACS Canto II Flow Cytometer (BD Biosciences) and analysed with InfinicytTM Software (Cytognos, Spain).
Other studies, such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), western blot, mitochondrial damage or enzyme activity assays are detailed in the supplementary files.
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