Cells were fixed with 4% paraformaldehyde (Sigma) for 20 minutes at room temperature, followed by addition of primary antibodies. Primary antibodies against SOX17 (1:200, R&D Systems), Ki67 (1:500, Cell Signaling), AFP (1:200, Dako), ALB (1:200, Dako), CD133 (1:100, Biolegend), and 8-OHdG (1:200, Abcam) were diluted in PBS with 0.3% BSA and 0.1% Triton X-100. Cells were incubated with appropriate primary antibodies at 4oC overnight, and then incubated with Alexa Flour 594 or 488 secondary antibodies (all of the Alexa Fluor Series from Invitrogen) at room temperature for 30 minutes. Finally, cells were counterstained with DAPI. Images were taken using the motorized Nikon Ti-E microscope and NIS-Elements software. Cell counting was performed with ImageJ software (http://imagej.nih.gov/ij /).
Cd133
Manufactured by BioLegend
Sourced in United States
CD133 is a cell surface glycoprotein that is commonly used as a marker for stem and progenitor cells. It is expressed on a variety of cell types, including hematopoietic stem cells, endothelial progenitor cells, and some cancer stem cells. CD133 plays a role in cell signaling and cell adhesion, but its specific biological functions are not fully understood.
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Lab products found in correlation
β catenin, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
8 ohdg, by Abcam (1 mentions)
Sox17, by R&D Systems (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Accutase, by Nacalai Tesque (1 mentions)
7 aad, by BD (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Wnt5a b, by Cell Signaling Technology (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Epidermal growth factor (egf), by Novus Biologicals (1 mentions)
Nestin, by BioLegend (1 mentions)
Ssea 1, by BioLegend (1 mentions)
Nanog, by BioLegend (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
15 protocols using cd133
1
Immunofluorescence Assay for Hepatic Cell Markers
2
Flow Cytometric Analysis of Cell Surface Markers
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The expression of surface proteins within the collected cells was determined by using flow cytometry (FC). Cells were dissociated with Accutase (Nacalai Tesque) and stained with CD24 (1555427; BD Biosciences), CD44 (103012; BioLegend, 338820; BioLegend), CD44v5 (L MCA1729; Bio-Rad), CD44v6 (MCA1730; Bio-Rad), CD44v7 (MCA1731; Bio-Rad), CD44v9 (LKG-M003; Cosmo Bio), CD133 (372808; BioLegend), and 7-AAD (372808; BD Biosciences). The relative fluorescence intensities were measured by using an SH800 cell sorter (Sony Corporation, Tokyo, Japan). A two-dimensionality reduction step was performed using t-distributed stochastic neighbor embedding (t-SNE) to visualize high-dimensional cell surface marker expression data in a low-dimensional space. Data were analyzed by using the FlowJo software, Version 10.2 (FlowJo).
3
Quantitative Protein Expression Analysis
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Primary antibodies N-cadherin, E-cadherin, Vimentin, β-catenin, p-GSK3β (S9), Dvl-2, Wnt5a/b, Notch1, CD44, p-Akt (ser473), Snail, and actin were purchased from Cell Signaling Technology (CST; Danvers, MA). CD44, CD133, and CD24 antibodies were purchased from Biolegend (BioLegend, Inc., San Diego, USA). Secondary Alexa 488-conjugated antibody and HRP conjugated antibody were purchased from CST.
4
Circulating Tumor Cells Profiling
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The extracted cells from 4 mL heparinized PB were stained with anti-CCL2 (monocyte chemoattractant protein-1) (eBioscience, San Diego, CA, United States), VEGF (Genature, Belgium), and EGF (Novus Biologicals, Littleton, CO, United States). This mixture was incubated at 4 °C. Staining was carried out by second layer antibodies, including fluorescein isothiocyanate for CCL2, R-phycoerythrin for VEGF, and phycoerythrin–indodicarbocyanine (Pe–Cy5) for EGF. Finally, by adding nuclear dye [4′,6-diamidino-2-phenylindole (DAPI)], the status of PE was assayed by the Leica EICA, DM RXA2-fluorescence microscope (Wetzlar, Germany). The CTCs were also assayed with cytokeratin 19 (KRT 19) (ScyTek, Logan, UT, United States), leukocyte common antigen [LCA cocktail (CD45) (BioCare Medical, Pacheco, CA, United States)], neuronal marker (NM) (ScyTek), and DAPI. Identification of brain CTCs was based on positive cells with DAPI/NM, and positive metastatic cells were confirmed through DAPI+/cytokeratin+/CD45-). The Manuel search was also performed to detect CTCs amongst the leukocytes. The CTCs were defined as nucleated cells by: (1) Expressing NM; and (2) Lacking CD45/positive KRT19. CD133 (Biolegend, San Diego, CA, United States), VEGF (Genature), and cyclin E (Zymed, San Francisco, CA, United States) were also assayed.
5
Phenotypic Profiling of Cancer Stem Cells
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Cells were stained with antibodies against CD133, SSEA1, Oct4, Nanog, Nestin (all from BioLegend, San Diego, CA, USA), MSI1 (BD Biosciences, San Diego, CA, USA), and BMI1 (R&D systems) to assess the changes in CSC-related gene expression. To assess the level of apoptosis, cells were stained with Annexin V plus PI or 7-AAD (all from BioLegend). Cells were analyzed by BD FACS Aria flow cytometer (BD Biosciences). Monoclonal antibodies against CASP3 (Cell Signaling Technology) were also used to assess the apoptotic cells.
6
Quantifying Circulating Endothelial Cells
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Blood samples were drawn at early morning, from the arms of hypertensive patients, in the recumbent position after 10 min of rest. Blood was collected from each patient into ethylenediaminetetraacetic acid (EDTA) anticoagulant and used for flow cytometric analysis. Multicolor flow cytometry analysis was performed according to the method published by Szpera-Goździewicz et al. (2017) (link) and modified by us elsewhere (Budzyń et al., 2018 (link)). The conjugated mouse anti-human monoclonal antibodies: CD34, CD146, CD45, and CD133 (BioLegend, London, United Kingdom) were used. The data were analyzed using BD FACSDiva software. The number of CD45 (−), CD34 (+), CD146 (+), CD133 (−) cells per 1,000,000 analyzed nucleated cells was defined as CECs, whereas CD45(−), CD34 (+), CD146 (+), and CD133 (+) cells as EPCs. The calibration of flow cytometry and the control of fluid stability were conducted each time before analysis. The results were expressed as the number of CECs and/or CEPCs per 4 ml of blood.
7
Protein and Phenotypic Analysis of Cell Lines
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CAF, MCF-10A, ZR-75-1, HCT116, QBC939, NCCIT, RBE, HUCCT1, ZJU-0826, and ZJU-1125 cells were washed thrice with ice-cold phosphate buffered saline (PBS), lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% sodium deoxyholate, 1% Nonide P-40, and 0.1% SDS with Pierce Protease Inhibitor Tablets (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstain A) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and separated by centrifugation. The protein concentration was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Antibodies against the following proteins were used: E-cadherin, N-cadherin, β-catenin, α-SMA, MUC1, CD146, SOX17, Vitamin D3 Receptor (VDR), pdx1, CD326, FoxA1/HNF3α, FoxA2/HNF3β, Nanog, GAPDH, and β-actin (all 1:1000, from Cell Signaling Technology, Danvers, MA, USA).
Flow cytometry was performed using a FACScan instrument (BD Bioscience, San Jose, CA, USA) and commercially available reagents for cells at passages 3–5 and 60–65. A panel of monoclonal antibodies was evaluated, including CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338 (Biolegend, San Diego, CA, USA). Antigen expression was determined based on a significant shift in staining compared to an isotype control.
Flow cytometry was performed using a FACScan instrument (BD Bioscience, San Jose, CA, USA) and commercially available reagents for cells at passages 3–5 and 60–65. A panel of monoclonal antibodies was evaluated, including CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338 (Biolegend, San Diego, CA, USA). Antigen expression was determined based on a significant shift in staining compared to an isotype control.
8
Antibody Sources for Stem Cell Research
9
CD44 and CD133 Expression in Caco-2 Cells
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CD44 (BD Pharmingen, San Diego, CA, USA) and CD133 (BioLegend, San Diego, CA, USA) were tested in this study. Caco-2 cells were washed with phosphate buffered saline (PBS, UniRegion Bio-Tech, Taiwan), and stained with markers for 30 minutes on ice at dark. The samples were then washed with PBS for 3 times, and analyzed by FACScalibur flow cytometer (Becton Dickinson).
10
Flow Cytometric Analysis of CD44 and CD133
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Surface markers, including CD44 (cat. no. 338804, BioLegend) and CD133 (cat. no. 372806, BioLegend), were assessed by flow cytometry. The cells were treated with trypsin and washed twice with PBS. Then, they were suspended with 100 μL PBS in order to reach a concentration of 1 × 107/mL. Subsequently, suspensions were administered with the antibodies at the standard concentration. After incubation in the dark for 30 minutes at 37°C, the suspensions were washed with PBS and resuspended by 200 μL of the same buffer. The samples were determined and sorted by FAC‐SCantoTM II flow cytometer (BD Biosciences), and the data were examined using FlowJo software (Tree Star).
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