Cells were dissociated with acutase and washed once with PBS. Cell surface levels were determined with antihuman antibodies CD133-FITC, (Miltenyi Biotec, Solothurn, Switzerland) diluted 1:300 in FACS buffer for 30 min in ice. All samples were analyzed on an FACS Melody (BD Biosciences, Allschwil, Switzerland) using the FlowJo software.
Cd133 fitc
Manufactured by Miltenyi Biotec
Sourced in Switzerland, Germany, United States
CD133-FITC is a fluorescently-labeled antibody that binds to the CD133 antigen. CD133 is a cell surface marker commonly used to identify and isolate stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Facsmelody, by BD (1 mentions)
Cellstripper, by Corning (1 mentions)
Cd133 apc, by Miltenyi Biotec (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Cd117 apc, by Miltenyi Biotec (1 mentions)
Cd44 fitc, by Miltenyi Biotec (1 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Cd44 pe, by Miltenyi Biotec (1 mentions)
Cd24 apc, by Miltenyi Biotec (1 mentions)
Cd105 apc, by Miltenyi Biotec (1 mentions)
Cd90 fitc, by Miltenyi Biotec (1 mentions)
Cd146 pe, by Miltenyi Biotec (1 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Facsjazz cell sorter, by BD (1 mentions)
Mission shrna, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
6 protocols using cd133 fitc
1
Flow Cytometric Analysis of CD133+ Cells
2
Isolation and Characterization of Cancer Stem Cells
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Cells were grown for 5–7 days in 3-D conditions. Spheroids were prepared into single cell suspensions using Cellstripper (Corning, Corning, NY, USA) and needle dissociation. Fluorescent-conjugated antibodies were purchased from Miltenyi Biotec CD44-FITC, CD117-APC, CD133-FITC, and CD133-APC (Table S2 ). ALDH activity was assessed using the AldeRed Kit (Millipore Sigma, Burlington, MA, USA) and AldeFluor Kit (Stem Cell Technologies, Vancouver, BC, Canada), according to manufacturer’s instructions. Sorting of positive and negative cells was performed on a BDFACS Melody cell sorter, directly into RNA lysis buffer and immediately processed for qRT-PCR studies. Data were analyzed using FlowJo 10.7.1 software.
3
Flow Cytometry Immunophenotyping of Cells
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For flow cytometry analysis, the cells were resuspended from the pellet in PBS from each sample and divided into different tubes. Each tube was individually incubated for 30 min at 4 °C with ECAD-APC, NCAD-PE, CD90-FITC, CD105-APC, CD24-APC, CD44-PE, CD133-FITC, and CD146-PE antibodies (all monoclonal) (Miltenyi Biotec, Bergisch Gladbach, Germany). After incubation, PBS was used to wash the cells and pelleted down. The labelled cells were resuspended in sheath fluid and analyzed with a flow cytometer (Attune NxT, Thermo Fisher Scientific, Waltham, MA, USA). At least for each samples 10,000 events are acquired. Percentage comparison was done with the degree of positive staining and the isotype controls. The median fluorescence intensities for each surface protein were also considered.
4
Quantifying CD133+ Cells in MNNG/HOS Cells
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To measure the proportion of CD133+ cells in single cell suspension of MNNG/HOS cells from the three groups cultured under different conditions, cells were digested with trypsin (Sigma-Alrdich; Merck KGaA), centrifuged at room temperature for 5 min at 200 × g, collected and washed twice with PBS. Cells were then incubated with CD133-FITC (1:100; Miltenyi Biotec, Inc.) at 4°C for 60 min in the dark, followed by washing twice with combining buffer (Beyotime Institute of Biotechnology). Finally, the labeled cells were analyzed NAVIOS flow cytometer (Beckman Coulter, Inc.).
5
Sorting and Culturing ESCC Cells
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The CSCs sorting were performed using the method described by Lonardo et al with minor modifications.20 Fresh ESCC tissues were cut into fine crumbles and digested 30 minutes in 37°C by 50 μg/mL collagenase. Cells were obtained after filtering the digestion products and adjusted to a concentration of 106 cells/mL in sorting buffer (1× PBS). Spheres were formed via culturing 3 × 103 primary ESCC cells, obtained from flow cytometer (Gallios, AV28109, California, USA) using CD133‐FITC (Miltenyi Biotec, 130104322, Cologne, Germany) and CD47‐PE (Biolegend, 323108, San Diego, CA) after staining (in 1× PBS 30 μL) at room temperature for 30 minutes. The media of serum‐free DMEM/F12 had to contain certain key component supplementing with B27 (1:50; Invitrogen, Carlsbad, California, USA), 20 ng/mL epidermal growth factor (EGF; 1:5000; R&D Systems), 20 ng/mL basic fibroblast growth factor (bFGF; 1:5000; R&D Systems) for a total of 7 days, allowing spheres to reach a size of >70 mm. For subsequent passaging, mature spheres, filter through 40‐μm cell strainers, were dissociated into single cells, and then recultured in media with anti‐CD47 (2 μg/mL; R&D systems, AF4670) or anti‐IgG control antibody (2 μg/mL; R&D, 5‐001‐A) for another 7 days.
6
Delineating Cancer Stem Cell Populations
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