For the characterization of BCSCs, cell lines MCF-7, MDA-MB-231 and SK-BR-3 were grown in 2D culture as monolayer and in 3D culture as mammospheres in suspension (only the positive subpopulation). These mammospheres were obtained by sorting with the Aldefluor Kit (see Section 4.3.1 .). After five days, cell lines were irradiated at 0, 2 and 6 Gy and characterization was performed 24 h after irradiation. The markers determined were the specific human antibodies anti-CD24 (APC) and anti-CD44 (PE) (Miltenyi Biotec, Bergisch Gladbach, Germany) and the enzyme ALDH1 activity was also detected using the Aldefluor Kit (StemCell Technologies, Vancouver, BC, Canada). Samples were analyzed on a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Three independent experiments were run in triplicate. The mean value for each experiment was calculated by averaging the triplicates (n = 3).
Anti cd44 pe
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD44-PE is a fluorescently-labeled monoclonal antibody that binds to the CD44 cell surface antigen. CD44 is a cell adhesion molecule involved in various cellular processes. The PE (phycoerythrin) fluorescent dye allows for the detection and analysis of CD44-expressing cells using flow cytometry.
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Lab products found in correlation
Anti cd24 apc, by Miltenyi Biotec (2 mentions)
Facscanto 2, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Igg1 pe vio770, by Miltenyi Biotec (1 mentions)
Igg1 apc, by Miltenyi Biotec (1 mentions)
Facsdiva software, by BD (1 mentions)
Igg1 pe, by Miltenyi Biotec (1 mentions)
Vantage se, by BD (1 mentions)
Anti cd133 phycoerythrin pe, by Miltenyi Biotec (1 mentions)
Anti cd14 fitc, by Miltenyi Biotec (1 mentions)
Anti cd133 apc, by Miltenyi Biotec (1 mentions)
Anti cd36 apc, by BD (1 mentions)
Anti cd11b pe, by BD (1 mentions)
Anti ssea4 fitc, by BioLegend (1 mentions)
Anti cd38 fitc, by Miltenyi Biotec (1 mentions)
Anti cd115, by BioLegend (1 mentions)
Anti cd56 apc, by BioLegend (1 mentions)
Anti cd146 fitc, by Miltenyi Biotec (1 mentions)
Anti cd138 pe, by Miltenyi Biotec (1 mentions)
Anti cd31 pe cy7, by Miltenyi Biotec (1 mentions)
5 protocols using anti cd44 pe
1
Characterization of Breast Cancer Stem Cells
2
Flow Cytometric Analysis of Cancer Stem Cell Markers
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To detect the expression of cancer stem cell markers, the following antibodies were used: anti-CD44-PE, anti-CD24-APC, anti-ESA-PE-Vio770, IgG1-PE, IgG1-APC, IgG1-PE-Vio770 (Miltenyi). Cells were harvested with trypsin-EDTA to produce a single cell suspension. The cells were pelleted by centrifugation, washed twice with PBS and incubated with the antibody for 10 min in the dark at 4℃. The respective isotype control antibodies were used at the same concentrations according to the manufacturer's instructions. After washing twice with PBS, samples were resuspended in 500 μl PBS and analyzed on a flow cytometer (FACSAriaII, USA). Cells were routinely sorted twice, and the cells were reanalyzed for the purity, which typically was >97%. Data were analyzed with BD FACS Diva software.
3
Flow Cytometric Analysis of CSC Markers
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For the flow cytometric analysis of CSC markers, cells were digested into single-cell suspensions and washed with PBS. Then, 1 × 106 cells were resuspended in 100 μL of PBS containing 0.5% BSA and 10 μL of fluorophore-conjugated primary antibody anti-CD133-phycoerythrin (PE) (Miltenyi Biotec, Germany) and anti-CD44-PE (Miltenyi Biotec, Germany) for 10 min in the dark at 4°C. Afterward, the tubes were removed by centrifugation and washed twice with 500 μL of PBS buffer. Next, cells were suspended in 200 μL of PBS and analyzed using a fluorescence-activated cell sorting (FACS) Vantage SE (BD Biosciences, Franklin Lakes, USA).
4
Multi-Marker Flow Cytometry Profiling
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Antibodies anti-CD31 PE-Cy7, anti-CD18 PE or APC, anti-CD51 FITC, anti-CD64 FITC, anti-CD14 FITC, PE or PerCP-Cy5.5, anti-CD66abce FITC, anti-CD34 VioBlue®, anti-CD38 FITC, anti-CD44 PE, anti-CD45 VioBlue® or APC-Vio770, anti-CD3 FITC or PerCP-Cy5.5, anti-CD20 FITC or PerCP-Cy5.5, anti-CD15 FITC, anti-CD16 PerCP-Cy5.5, anti-HLA-DR VioBlue®, anti-CD86 VioBlue®, anti-CD80 VioBlue®, anti-CD90 FITC and PerCP-Cy5.5, anti-CD133 APC, anti-CD138 PE, anti-CD140a PE, anti-CD146 FITC, and anti-MSCA-1 PE were from Miltenyi Biotec. Anti-STRO-1 AlexaFluor® 647, anti-CD73 PE-Cy7, anti-CD105 PerCP-Cy5.5, anti-CD140b PE, anti-CD56 APC, anti-CD309 PerCP-Cy5.5, anti-CD115, anti-CD144 PE, anti-SSEA-3 FITC, and anti-SSEA-4 FITC were from BioLegend (San Diego, CA). Anti-CD271 PE, anti-CD11b PE, anti-CD11c PE, and anti-CD36 APC were from BD Biosciences (San Jose, CA).
5
Flow Cytometry Analysis of ASCs
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ASCs at passage three were analyzed by flow cytometry with a CytoFLEX flow cytometer (Beckman Coulter, Fullerton, CA, USA), collecting at least 10,000 events. Antibodies used to confirm ASC phenotype [15 (link)] were: anti-CD44-PE (Cat# 130-110-293), CD90-FITC (clone REA897), CD105-PerCP-Vio700 (clone REA794), CD45-PE Vio770 (clone REA747) (Miltenyi Biotec, Bergisch Gladbach, Germany). Doublets were removed from analysis gating events on FSC-H and FSC-A plot.
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