The expression of NSCLC cell markers and molecules involved in the adenosinergic pathways were evaluated by flow cytometry. Adherent cells were detached through trypsin and spheres were dissociated using accutase solution (Sigma-Aldrich, St. Louis, MO, USA), then the single-cell solution was washed in staining buffer (PBS 1X + 0.5% BSA + 2mM EDTA) and incubated for 20 min at RT with the following anti-human antibodies: PE-CD133/1 (Miltenyi Biotech, Auburn, CA, USA), APC-CXCR4 (Miltenyi Biotech, Auburn, CA, USA), FITC-CD73, and PE-PC1 (kindly provided by the Malavasi Lab.), CD38 (Miltenyi Biotech, Auburn, CA, USA), CD26 (eBioscience, San Diego, CA, USA), and CD39 (BD Biosciences, Franklin Lakes, NJ, USA). Isotypic controls and unstained samples were used to set the negative control. Data were acquired using a MACsQuant 10 cytometer and analyzed through MACsQuantify software (Miltenyi Biotech, Auburn, CA, USA).
Cd133 1 pe
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
The CD133/1-PE is a fluorochrome-conjugated monoclonal antibody that specifically binds to the CD133 antigen, also known as Prominin-1. CD133 is a pentaspan transmembrane glycoprotein that is expressed on various types of stem and progenitor cells. The PE fluorochrome allows for the detection and analysis of CD133-positive cells using flow cytometry or other immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Macsquant10 cytometer, by Miltenyi Biotec (1 mentions)
Accutase solution, by Merck Group (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Cxcr4 apc, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Cd29 apc clone ts2 16, by BioLegend (1 mentions)
Anti mouse igg1 alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions)
Facs aria sorp cytometer, by BD (1 mentions)
Trypsin, by Lonza (1 mentions)
Cd44 fitc, by Immunotools (1 mentions)
Cd34 pe, by BD (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd49d pe, by BD (1 mentions)
Cd57 fitc, by BD (1 mentions)
Cd13 pe, by BD (1 mentions)
Cd44 fluorescein isothiocyanate fitc, by BD (1 mentions)
10 protocols using cd133 1 pe
1
Adenosinergic Pathway Profiling in NSCLC
2
Multiparameter Flow Cytometry for Disseminated Tumor Cells
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List of anti‐human antibodies: PE‐CD133/1 (Clone AC133, Miltenyi Biotec), APC‐CXCR4 (Clone 12G5), FITC‐CD44 (Clone L178), PE‐CD166 (Clone 3A6) (all from BD Bioscience) and CD29‐APC (clone TS2/16, BioLegend) were incubated with cells for 30 minutes at 4°C.
Integrin β1 antibodies: anti‐mouse CD29 (Clone 9EG7‐550531 recognizing the active form of integrin β1), anti‐human CD49b (Clone 12F1‐555668), anti‐human CD49a (clone SR84 559 594) (all from BD Bioscience) were incubated with cells for 1 hour at room temperature (RT), then washed and incubated with secondary antibody (anti‐rat IgG2 Alexa Fluor 488 and/or anti‐mouse IgG1, Alexa Fluor 647, Thermo Fisher), for 30 minutes at RT.
Prior to FACS analysis, samples were incubated with 7‐AAD viability staining solution (10 μL/tube) (eBioscience) to exclude dead cells.
FACSCalibur and FACSCanto cell analyzers (Becton Dickinson) were used for data acquisition and FlowJo software V10 was used for data analysis.
LT73 CD133neg cell line was generated by sorting CD133 negative cells from the parental LT73 cell line using FACSAria cell sorter (BD Biosciences), as already reported.17 Human disseminated tumor cells (DTC) were quantified in dissociated murine lung tissue, as 7AAD‐ live cells negative for anti‐mouse MHC class I (eBioscience), and their relative content of CD133+ CXCR4+ MIC was evaluated, as already detailed.17
Integrin β1 antibodies: anti‐mouse CD29 (Clone 9EG7‐550531 recognizing the active form of integrin β1), anti‐human CD49b (Clone 12F1‐555668), anti‐human CD49a (clone SR84 559 594) (all from BD Bioscience) were incubated with cells for 1 hour at room temperature (RT), then washed and incubated with secondary antibody (anti‐rat IgG2 Alexa Fluor 488 and/or anti‐mouse IgG1, Alexa Fluor 647, Thermo Fisher), for 30 minutes at RT.
Prior to FACS analysis, samples were incubated with 7‐AAD viability staining solution (10 μL/tube) (eBioscience) to exclude dead cells.
FACSCalibur and FACSCanto cell analyzers (Becton Dickinson) were used for data acquisition and FlowJo software V10 was used for data analysis.
LT73 CD133neg cell line was generated by sorting CD133 negative cells from the parental LT73 cell line using FACSAria cell sorter (BD Biosciences), as already reported.
3
Flow Cytometry Analysis of CD133 Expression
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Cell surface antigen expressions were analyzed using flow cytometry. CD133/1-PE (Miltenyi Biotec) and mouse IgG1-PE isotype control antibody (Miltenyi Biotec) were used according to manufacturer's instructions. MGG8 was incubated with these antibodies for 10 min at 4°C and then washed and analyzed using a MACSQuant Analyzer (Miltenyi Biotec).
4
Flow Cytometric Detection of CSC Markers
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Detection of CSC markers was performed using CD44-FITC/APC (Becton Dickinson, Auburn, CA, USA), ESA-FITC (StemCell Technologies, Vancouver, BC, Canada), ALDH1-PE (Miltenyi Biotec, Auburn, CA, USA), and CD133/1-PE (Miltenyi Biotec) antibodies. Approximately 1 × 106 cells were distributed into tubes containing PBS with 2% fetal bovine serum and kept on ice for 10 min. Antibodies were added to cell suspensions and incubated on ice in the dark for 30 min. Cells were washed and re-suspended in 500 mL FACS buffer and analyzed using a flow cytometer (BD Biosciences, CA, USA).
5
Cell Membrane Staining and FACS Analysis
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A single-cell suspension of cultured cell lines was obtained by trypsinization (0.25% trypsin, Lonza) at 37°C for 2–3 min. For cell membrane staining cells were resuspended in Hank's balanced salt solution w/o Ca2+/Mg2+ (HBSS), 2% FBS, 10 mmol/L HEPES pH 7.4 buffer (106cells/100 μL/test) and incubated with LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen, Life Technology Ltd.; 1 μg/mL) and appropriate pre-conjugated antibodies (CD133/1-PE, Miltenyi Biotec, Bergisch Gladback, Germany, 10 μL/test; CD15-PE Immunotools, Friesoythe, Germany, 10 μL/test; A2B5-APC Miltenyi Biotec, 10 μL/test; CD44-FITC, Immunotools, 10 μL/test) 30 min in the dark. Data acquisition was performed with a FACS Aria™ SORP cytometer (BD Biosciences, San Jose, CA) and flow graphs were prepared with the FlowJo software (Tree Star, Inc., Ashland, OR).
6
Multiparametric Characterization of HUCB and Bone Marrow Cells
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Antibodies against the human antigens CD13-PE, CD31-PE, CD34-PE, CD44-fluorescein isothiocyanate (FITC), CD49d-PE, CD57-FITC, CD73-PE, CD90 (Thy-1)-pure, and CD105 (Endoglin)-pure were purchased from BD Biosciences (San Jose, CA), CD45-FITC was from Immunotech (Marseille, France), and CD133/1-PE, and CD271(p75NTR)-FITC, -PE, -APC were obtained from Miltenyi Biotec. For each antibody expression assessment, a total of 1 × 105 MNCs from HUCB and human bone marrow were resuspended in 100 μl PBS containing 0.5% BSA and 200 mM EDTA, incubated with rabbit serum for blocking nonspecific binding. The cells were then incubated with primary antibodies for 30 min on ice. Binding of unconjugated anti-CD90 and CD105 was detected by secondary staining with PE-conjugated anti-mouse IgG antibody (BD Biosciences). FITC- and PE-conjugated mouse IgGs were used as the control isotype at the same concentration as specific primary antibodies. The fluorescence intensity of the cells was evaluated by flow cytometry (FACSCalibur, BD Biosciences), and data was analyzed with CellQuest software (BD Biosciences).
7
Flow Cytometric Analysis of CD133+ Cells
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Trypsinized cells were centrifuged and resuspended in FACS buffer (PBS, pH 7.2, containing 0.5% fetal bovine serum and 2 mM ethylenediaminetetraacetic acid). Phycoerythrin (PE)-conjugated immunoglobulin G (isotype control), and CD133/1-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to each group with an FcR blocking reagent (Miltenyi Biotec) and incubated in the dark for 10 min at 4°C. After washing, the cells were resuspended in FACS buffer for analysis with a BD FACS Aria III (BD Bioscience, San Jose, CA, USA).
8
Evaluating Autophagy and Apoptosis in Cancer
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Gemcitabine, Paclitaxel and Cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). ALDEFLUOR assay was from StemCell Technologies (Durham, NC, USA). CD133/1PE (used for flow cytometry) was from Miltenyi Biotec (Bergisch Gladbach, Germany). Monoclonal antibodies: anti-ITCH antibody was from BD Transducion Laboratories (San Jose, CA, USA), anti-p62 SQSTMI from Santa Cruz Biotechnologies (Dallas, Texas, USA), anti-PARP1 from ENZO (New York, NY, USA), anti-actin from Sigma-Aldrich (St. Louis, MO, USA); polyclonal antibodies: anti-LC3 was purchased from Sigma-Aldrich and anti-caspase3 from Cell Signaling (Danvers, MA, USA). Secondary anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase were from Bio-Rad (Hercules, CA, USA).
9
CD133 and CXCR4 Expression Analysis
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Cells were washed twice in ice-cold PBS and stained with CD133/1-PE (Miltenyi Biotec, CA, USA), and APC-conjugated anti-CXCR4 antibody (BD Biosciences, 560936, San Diego, CA, USA). The stained cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson).
10
Flow Cytometry Analysis of Cell Surface Markers
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For flow cytometry analysis, the cell lines were harvested wit 0.25% trypsin, 0.02% EDTA (Sigma Aldrich). The cells were resuspended in cell culture media and were then counted and washed in PBS with 2% FCS and 5 mM EDTA and collected by centrifugation. The cells were blocked with FcR-Blocking reagent human (Miltenyi Biotec) for 10 min on ice. Afterwards, the cells were stained with CD133/1-PE (Miltenyi Biotec) or CD44 (AC-CD44-PE, Miltenyi) at 4°C for 30 min in the dark. Following antibody labeling, the cells were washed twice with PBS with 2% FCS and 5 mM EDTA. Finally, the cells were resuspended in PBS with 2% FCS and 5 mM EDTA. Data were acquired on a FACSCanto II (BD Biosciences, San Jose, USA) and analyzed with Diva-BD software.
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