For the spheroid formation assays, Huh7 cells were treated with MCL (30 µM) or DMSO for 8 hr and further incubated with fresh medium for 24 hr. Subsequently, 1000 cells were collected, suspended in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL basic FGF and B27 supplements, and seeded on a Costar® Ultra Low Attachment 6-well plate (Corning) for 14 days. Spheroids were counted to measure the spheroid-forming index when larger than 100 µm. To evaluate the expression of the stemness marker, HCC cells were preformed to fluorescence-activated cell-sorting cytometry (FACS) analysis. After treatment of MCL as above, 1×106 cells were collected, washed, incubated for 30 min at 4 °C with respective fluorescence-conjugated antibodies including CD44-FITC, EpCAM-FITC, and CD133-PE (BD, Biosciences, San Jose, CA, USA). The appropriate fluorochrome-conjugated, isotype-matched antibodies were used as a control to establish background staining. Samples were acquired on a FACS Forte and data were analyzed with DIVA software (BD, Biosciences, San Diego, CA).
Cd133 pe
Manufactured by BD
Sourced in United States
CD133-PE is a fluorescent-labeled antibody used for the detection and isolation of CD133-positive cells in flow cytometry and cell sorting applications. CD133, also known as prominin-1, is a surface marker expressed on various stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd44 fitc, by BD (5 mentions)
Cd34 pe, by BD (2 mentions)
Cd29 pe, by BD (2 mentions)
Cd34 apc, by BD (2 mentions)
Cd45 percp, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Cd44 pe cy7, by BD (2 mentions)
Epcam fitc, by BD (1 mentions)
Costar ultra low attachment 6 well plate, by Corning (1 mentions)
Diva software, by BD (1 mentions)
Cd117 apc, by BD (1 mentions)
Alexafluor 546 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsrii system, by BD (1 mentions)
Ros detection reagents fitc, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Cd90 pe, by BD (1 mentions)
Cd106 fitc, by BD (1 mentions)
Cd117 pe, by BD (1 mentions)
Cd147 fitc, by BD (1 mentions)
Cd105 fitc, by BD (1 mentions)
15 protocols using cd133 pe
1
Stemness Evaluation of Huh7 Cells
2
Characterization of CSC Phenotype in Spheres
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For further characterization of CSC phenotype of sphere forming SP cells, flow cytometry was used. Spheres were enzymatically dissociated into single cell suspension (1X106). Cells were washed twice with PBS and were stained with CD133-PE and CD117-APC (550412; BD Pharmingen) for 45 minutes at 4°C. Respective mouse IgG isotype were used as control. After incubation cells were washed with PBS and stained with fluorochrome conjugated anti mouse IgG Alexa Fluor 546 (invitrogen) at a dilution of 5 μl of antibody per 106 cell and re incubated further in dark on ice for 30 minute, washed, re-suspended in HBSS and stained by Propidium iodide (1 μg/ml). Analysis and sorting were performed on FACS Aria II using Diva Software.
3
Endothelial Progenitor Cell Characterization
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An endothelial cell marker combined with a stem cell marker CD34+/CD133+ was used to identify EPC as previously described (30 (link)). Murine blood cells were harvested after sacrifice to detect EPC and mononuclear cell intracellular ROS formation via flow cytometry by using ROS Detection Reagents-FITC (cat. no. D399; Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (31 (link)). After removing red blood cells (RBCs) using 1X RBC lysis buffer (cat. no. 00433357; Thermo Fisher Scientific), a total of 50,000 cells in each sample were incubated with 5 µg/ml ROS Detection Reagents-FITC for 10 min at 37°C. BD™ LSRII (BD Biosciences) at a wavelength of 525 nm was used to calculate the positively fluorescent cells for intracellular ROS detection. For EPC measurement, cells were incubated with anti-mouse CD34-AF700 (cat. no. 560518; BD Biosciences) and CD133-PE (cat. no. 12-1331-82; eBioscience; Thermo Fisher Scientific, Inc.) as previously described (21 (link)), then incubated with 5 µg/ml ROS Detection Reagents-FITC for 10 min at 37°C. Labeled cells were washed twice with PBS and suspended in warm PBS for analysis by flow cytometry on a BD™ LSRII system (BD Biosciences). All antibodies were diluted 1:100.
4
Phenotypic Characterization of Adherent AFCs
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Adherent AFCs were separated by trypsin treatment and fixed in 75% ethanol overnight at 4°C. The cells were filtered through a 40 μm mesh and resuspended in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 0.1% sodium azide]. Directly-conjugated isotype control antibodies, IgG-fluorescein isothiocyanate (FITC) and IgG-phycoerythrin (PE; BD Pharmingen, San Diego, CA, USA), were used as controls to identify the background cells. A total of ~5×105 cells were incubated at 4°C for 40 min with each of the following FITC- or PE-conjugated antibodies (BD Pharmingen): CD133-PE, CD117-PE, CD34-PE, CD105-FITC, CD106-FITC, CD29-PE, CD44-FITC, CD147-FITC and CD90-PE. The cells were subsequently washed in FACS buffer. The antibody-labeled cells were analyzed using a BD FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo version 7.2.5 software (TreeStar, Inc., Ashland, OR, USA). The cell count experiments for the flow cytometry assays were performed in triplicate.
5
Multiparametric Flow Cytometry for Tumor Stem Cells
6
Identification of Gastric and Breast Cancer Stem Cells
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Human gastric cancer cells (AGS and MKN1) were dissociated into single cells, washed with PBS, and stained with fluorescent antibodies for CD133-PE (BD Biosciences, Franklin Lakes, New Jersey) and CD44-FITC (BD Biosciences, Franklin Lakes, New Jersey). To determine the effect of ROS levels on M-and E-BCSCs in breast cancer cell lines, MCF7 cells were incubated with antibodies against CD24-PE (BD Biosciences, Franklin Lakes, New Jersey) and CD44-FITC. Content of ALDH+E-BCSCs was determined by Aldefluor assay (StemCell Technologies) per manufacturer's instructions. The cells were sorted using a BD FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
7
Immunophenotyping and Drug Screening
8
Profiling Stem Cell Markers by Flow Cytometry
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Cells were washed in phosphate buffered saline (PBS) and collected by centrifugation. The cells were stained with primary antibodies including CD44-FITC (560977, BD Pharmingen), CD24-Alexa 647 (56144, BD Pharmingen), c-Met-Alexa 647 (566014, BD Pharmingen), CD326-BB515 (565398, BD Pharmingen), CD133-PE (VII 70485, BD Pharmingen) at 4 °C for 30 min. After washing with PBS twice and re-suspended in 800 µL PBS, the cells were filtered through a 70 mm nylon mesh and carried out on a flow cytometer (BD Biosciences, Heidelberg, Germany). The viable and single cells were gated for analyses. BD CellQuest pro (BD Biosciences) and FlowJo (BD Bioscience) software were used to analyze the data.
9
Flow Cytometry of Cell Surface Markers
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Cells were harvested at 48 hpi (MOI = 0.005) by trypsinization and washed twice with PBS. Cells were stained with fluorochrome-conjugated antibodies: Live/Dead fixable near-IR stain (L34976A; Invitrogen), CD44-APC (# 560890) and CD133-PE (# 566594) purchased from BD Biosciences (Franklin Lakes, NJ). Cells were stained for 30 min at RT as described previously.41 (link) After two washes, cells were resuspended in 200 μL PBS. Samples were analyzed on a CytoFlex flow cytometer (Beckman Coulter, Brea, CA) and FlowJo software was used for analysis.
10
Quantification of Circulating Endothelial Cells
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Circulating endothelial cells (CEC) were measured as per the protocol described by Duda et al. [37 (link)]. Briefly, patient blood was collected prior to MRI imaging using tubes containing EDTA. Fluorescently labelled antibodies were purchased from BD Pharmingen: CD31-FITC, CD34-APC, CD133-PE, CD45-PerCP, Fluorescently labeled isotype-matched IgG1 antibodies, and VEGFR2 (KDR)-PE. Cells from the buffy coat were fixed, labeled using the above listed antibodies, then sorted using the FACSCalibur flow cytometer (Becton Dickinson).
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