Ovarian cancer spheroids cultured in microfluidic chip were dissociated into single cells and double-labeled with mouse anti-human CD117-PE and mouse anti-human CD44-FITC (BD Biosciences, San Jose, CA) following the manufacturer’s instruction. Percentage of CD117+/CD44+ cells was analyzed using BD FACSAriaTM III (BD Biosciences).
Fitc mouse anti human cd44
Manufactured by BD
Sourced in United States, Italy
The FITC mouse anti-human CD44 is a monoclonal antibody that binds to the CD44 antigen expressed on the surface of various cell types, including hematopoietic and non-hematopoietic cells. The antibody is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate), allowing for the detection and analysis of CD44-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Facsaria 3, by BD (3 mentions)
Cellquest software, by BD (3 mentions)
Pe mouse anti human cd24, by BD (3 mentions)
Pe mouse anti human cd90, by BD (3 mentions)
Fitc mouse anti human cd45, by BD (3 mentions)
Pe mouse anti human cd73, by BD (3 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Pe mouse anti human cd34, by BD (2 mentions)
Pe mouse anti human hla dr, by BD (2 mentions)
Pe mouse anti human cd29, by BD (2 mentions)
Fitc mouse anti human cd105, by BD (2 mentions)
Fitc mouse anti human hla abc, by BD (2 mentions)
Fitc mouse anti human cd31, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cd29 apc, by BD (1 mentions)
Cd34 fitc, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Cd14 pe, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd11b pe, by BD (1 mentions)
20 protocols using fitc mouse anti human cd44
1
Ovarian Cancer Spheroid Phenotyping
2
Cell Surface Antigen Immunofluorescence
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Immunofluorescence staining of cell surface antigens was performed by incubating cells for 30 min at 4 °C in the dark with mouse anti-human CD44-FITC, CD34-FITC, human leukocyte antigen (HLA)-DR-FITC, CD105-PE, CD14-PE, CD11b-PE, CD29-APC and CD45-APC Abs (all from BD Bioscience, San Jose, CA). Cells incubated in the absence of antibodies were used as negative controls. Cells were then washed 3 times with PBS, fixed with 1% (w/v) formaldehyde in PBS and subjected to flow cytometry using a FACSCalibur analyzer and CellQuest software (BD Biosciences).
3
CD44 Expression Profiling in H1299 Cells
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H1299 cells were stained with mouse IgG-FITC (BD Biosciences) or mouse anti-human CD44-FITC (BD Biosciences) for 20 min on ice. Labeled cells were sorted by FACSAria III (BD Biosciences). IgG staining was used as negative control.
4
Multiparametric Flow Cytometry Analysis
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Treated and untreated cells were detached by trypsin, counted and washed in 0.1% BSA in PBS. At least 500,000 cells were incubated with fluorescent-labeled monoclonal antibodies or respective isotype controls (1/10 diluted 4 °C for 30 min in the dark). After washing steps, the labeled cells were analyzed by flow cytometry using a BD FACS Aria III (Becton & Dickinson, Mountain View, CA, USA). The antibodies used were mouse anti-human CD133/2 PE (Miltenyi Biotec S.r.l. Calderara di Reno, Bologna, Italy), mouse anti-human CD29 PerCP-Cy5.5 (BD Pharmingen, Buccinasco, Milan, Italy), mouse anti-human CD44 FITC (BD Pharmingen, Buccinasco, Milan, Italy), and mouse anti-human CD324 PE (BD Pharmingen).
For intracellular and nuclear staining of vimentin, osteocalcin, OCT4 PE, Sox2 FITC and Nanog PerCP-Cy5.5, cells were processed using the Caltag Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer’s guidelines. Isotypes were used as controls. All data were analyzed using a Diva software 6.6.
For intracellular and nuclear staining of vimentin, osteocalcin, OCT4 PE, Sox2 FITC and Nanog PerCP-Cy5.5, cells were processed using the Caltag Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer’s guidelines. Isotypes were used as controls. All data were analyzed using a Diva software 6.6.
5
Phenotypic Characterization of Melanoma Cells
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Cells were grown at 80% confluence, fixed in 4% PFA and stained with 0,15% crystal violet. Cell images were captured using an inverted microscope (Leica, DMI6000). Cells were stained following incubation for 30 min at +4°C with the following antibodies (5 µl/ml): mouse anti-human CD90 PE-Cy5, mesenchymal marker (BD Biosciences, Franklin Lakes, NJ, USA), human E-cadherin (CD324) APC, human CD133/2 (293C3)-PE (both from Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany), mouse anti-human CD20 FITC and mouse anti-human CD44 FITC (both from BD Biosciences). After being washed with PBS, the samples were analyzed by FACS. The fold-change of the mean fluorescence intensity (MFI) values was used to show the differences in surface marker expression between drug-sensitive and -resistant melanoma cells. It was calculated from 3 independent experiments.
6
Isolation and Characterization of CD44-Expressing Cells
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Human K562 CML cell line was obtained from American Type Culture Collection (Manassas, VA). CD44high K562 cells were established during isolation of imatinib-resistant K562 cells after treatment with increasing concentrations of imatinib, and were stable in complete medium without imatinib. The profile of cell surface CD44 expression was carried out on both cells and gated using mouse anti-human CD44-FITC (BD Biosciences, San Jose, CA, USA).We also used CD44+ HCT-15 cells with high CD44 expression and CD44- HCT-15 cells with low CD44 expression isolated from human colon cancer cell line HCT-15 29 (link). Cells were maintained in RPMI medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v⁄v) heat-inactivated FBS (Gibco BRL, Life Technologies, Carlsbad, CA, USA), 100 U⁄ml penicillin and 100 mg⁄ml streptomycin (Sigma-Aldrich, St.Louis, MO, USA) in a 5% CO2 humidified incubator at 37°C. 17-AAG and AUY922 were purchased from Enzo Life Sciences Inc. (Farmingdale, New York, USA) and Selleck Chemicals (Houston, TX, USA), respectively. EX527 was purchased from BioVision Inc. (Milpitas, CA, USA). Amurensin G, a natural SIRT1 inhibitor, was supplied Dr. Oh (Seoul National University, Seoul, Korea) as described previously 30 (link).
7
Comprehensive Protein Expression Analysis
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Antibodies that recognize c-Met, p-c-Met (Tyr1234/1235), ERK1/2, p-ERK1/2, AKT, p-AKT, E-cadherin, and vimentin were purchased from Cell Signaling Technology. Antibodies against PLXDC2 and ELK1 were ordered from Proteintech and Abcam, respectively. The c-Met inhibitor SU11274 and β-Actin antibody were obtained from Sigma-Aldrich. The secondary antibodies were purchased from Invitrogen. PE mouse anti-human CD133 and FITC mouse anti-human CD44 were obtained from BD Biosciences and used for flow cytometry. The c-Met inhibitor foretinib and crizotinib, AKT inhibitor AZD5363, and ERK1/2 inhibitor SCH772984 were obtained from SelleckChem. Cell viability was measured by alamarBlue Cell Viability Reagent (Thermo Fisher Scientific) following the indicated treatments. Western blotting, wound healing assays, cell proliferation, and colony formation assays were carried out as described previously (15, 16 (link)).
8
Disulfiram and Copper Compounds in NSCLC
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The non-small cell lung cancer (NSCLC) A549 (CCL-185) and H23 (CRL-5800) cell lines were purchased from ATCC (Middlesex, UK). Copper chloride (CuCl2, Cu), disulfiram (DS), diethyldithiocarbamate (DDC), S-methyl-N,N-diethyldithiocarbamate (S-Me-DDC), tetramethylthiuram disulfide (TMDS), 2-hydroxy-dithiobenzoic acid (HDTA), 4-imidazoledithiocarboxylic acid (IDTA), 2,4,6-Trimercaptotriazine (TMT) and poly-2-hydroxyethyl methacrylate (poly-HEMA) were purchased from Sigma (Dorset, UK). Bis(N,N-diethyldithiocarbamate)-copper(II) (Cu-DDC) was from Santa Cruz (Dallas, TX, USA). Annexin V kit was from Roche Applied Sciences (Burgess Hill, UK). Fc OxyBURST Assay Reagents was purchased from Invitrogen, Molecular Probes (Waltham, MA, USA). ALDEFLUOR kit was from StemCell Tech (Durham, NC, USA). FITC mouse Anti-Human CD44 from BD Biosciences (Oxford, UK).
9
Differentiation and Characterization of CR-CSC
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2 × 105 CR-CSC cell lines Sa41, Sa47, Re121, and U11 were differentiated in tissue culture plates (six-well) for seven days in CSC medium supplemented with FBS 10% in the absence of growth factors. Following adhesion, indicative of differentiation, CR-CSC were eventually treated with 2 mM NaB, 20 mM NH4Cl, 5 µM GW4869, 2 mM NaB + 20 mM NH4CL, 2 mM NaB + 5 µM GW4869 and after 48 h tested for the expression of CD44 and CD133 by flow cytometry. Briefly, the cells were collected, washed with PBS and incubated with anti-CD44 (FITC mouse anti-human CD44, BD PharmigenTM, Milano, Italy, 1:20) and anti-CD133 (PE mouse anti-human CD133/2, Miltenyi Biotec, Bologna, Italy, 1:20) antibodies at 4 °C for 1 h. Cells were then washed with PBS, collected by centrifugation and analyzed by Cytoflex S (Beckman Coulter, Indianapolis, IN, USA) and with CytExpert Software (Beckman Coulter Indianapolis). Negative controls were obtained by incubating the cells with PE and FITC Human IgG1 Isotype Control Antibody (Biolegend, San Diego, CA, USA). The assays were repeated at least three times and gave similar results. The data reported are the results of a representative experiment.
10
Delineating Cancer Stem Cell Markers
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To delineate cancer stem cell markers CD44 and CD133 expression in HCC cells, flow cytometry was used to detect the levels of the cell surface proteins. Cells were harvested and incubated with FITC mouse anti-human CD44 (BD PharmingenTM, #555478) and PE anti-human CD133 (BioLegend, #372804) in 4 °C for 30 min. The measurement was performed using a FACS Calibur flow cytometer (BD Biosciences).
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