The single-cell suspensions were incubated with phycoerythrin (PE) anti-human CD133 antibody (1:50, Cat. 372803, BioLegend, USA) and fluorescein isothiocyanate (FITC) anti-human CD326 antibody (1:50, Cat. 324203, BioLegend, USA) in staining solution containing 3% bovine serum albumin (BSA) for 15 min at 4°C according to the manufacturer’s instructions. Negative controls were stained with antibodies of isotype control. The cells were then washed and resuspended in pre-cooled PBS, and filtered through a 40 μm cell strainer to obtain single-cell suspension. Cell analysis was performed using a BD Accuri C6 Plus (BD Biosciences, USA), and cell sorting was performed using a BD FACS Aria ш (BD Bioscience, USA). Data were analyzed using FlowJo 10.5.3 software (FlowJo, LLC) and only living and single cells were gated out for population analysis using a PI gate and side and forward scatter gates. All experiments were repeated thrice.
Pe anti human cd133 antibody
Manufactured by BioLegend
Sourced in United States
PE) anti-human CD133 antibody is a lab equipment product that binds to the CD133 antigen, a cell surface glycoprotein commonly used as a marker for stem and progenitor cells. This antibody is conjugated with the fluorescent dye Phycoerythrin (PE) for detection and analysis purposes.
Automatically generated - may contain errors
4 protocols using pe anti human cd133 antibody
1
Isolation and Characterization of CD133+CD326+ Cells
2
CD133 and ALDH1 Expression Analysis
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To evaluate CD133 expression by flow cytometry, cells were harvested, washed with Cell Staining Buffer (cat. no. 420201; Biolegend, Inc.), and then incubated with PE anti-human CD133 antibody (1:200; cat. no. 372803; Biolegend, Inc.) for 15–20 min on ice in the dark. Cells were then washed and suspended in Cell Staining Buffer (at room temperature for 5 min) for analysis. The data acquired on the Guava easyCyte 8HT Base System were analyzed using the InCyte software. ALDH1 enzymatic activity was assessed using an Aldefluor kit (cat. no. 01700; STEMCELL Technologies Inc.) according to the manufacturer’s instructions. Cells suspended in the Aldefluor assay buffer were incubated with ALDH enzyme substrate, BODIPY-aminoacetaldehyde (BAAA; 1:200) for 30–60 min at 37 °C. As a control for baseline fluorescence, cells were also treated for 30–60 min at 37 °C with the ALDH inhibitor, diethylaminobenzaldehyde (DEAB at 1:100 dilution). Fluorescence was detected using the Guava easyCyte 8HT Base System and analyzed using the InCyte software. Statistical significance was determined by the paired Student’s t test or one-way ANOVA test.
3
Quantification of CD133+ Osteosarcoma Cells
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Osteosarcoma cells were harvested with fresh 0.25% trypsin solution (Sigma–Aldrich) and suspended in phosphate-buffered saline (PBS). Cells were blocked on ice for 15 min and subsequently labelled with PE anti-human CD133 antibody (BioLegend) for 60 min. The cells were then washed twice with PBS and maintained on ice until analysis. Expression levels were determined by flow cytometry (FACS Calibur, BD Bioscience, USA) and data were analyzed using WinMDI software (Scripps Research Institute, La Jolla, CA,USA).
4
Colon Cancer Cell Cycle Analysis
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Human colon cancer cells, serum starved for three days followed by treatments, fixed (ethanol), permeabilized (Triton-x 100), and stained for DNA (Propidium Iodide) were used for flow cytometry analysis (Cytek Aurora, 4 laser system). The experiment was performed four independent times and data were analyzed using SpectroFlo and Modfit software. Flow cytometry analysis was also performed using conjugated phycoerythrin (PE) anti-human CD133 antibody (Biolegend, San Diego, CA) for the colon cancer stem cells population. The experiment was performed three independent times and data were acquired using BD FACS Aria III and results were analyzed by BD FACSDiva software.
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