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Apc conjugated cd44 antibody

Manufactured by BD
Sourced in United States

The APC-conjugated CD44 antibody is a specific antibody that binds to the CD44 cell surface antigen and is labeled with the fluorescent dye allophycocyanin (APC). CD44 is a transmembrane glycoprotein involved in cell-cell interactions, cell adhesion, and migration. The APC-conjugated CD44 antibody can be used for the identification and analysis of CD44-expressing cells in flow cytometry applications.

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5 protocols using apc conjugated cd44 antibody

1

Cell Surface Marker Analysis by Flow Cytometry

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For cell-surface analysis, cells were harvested by dissociation using 0.05% trypsin/EDTA. A total of 1 × 106 cells were resuspended in 200 μL PBS with 1% FBS, incubated with antibodies at the recommended concentrations at 4 °C for 30 min, and then detected by flow cytometry (BD FACSCanto II, BD Biosciences, USA). Data were analyzed using FlowJo software. The antibodies used in flow cytometry assay were as follows: APC-conjugated CD163 antibody (333610, Biolegend), PE-conjugated CD206 antibody (321106, Biolegend), APC-conjugated CD44 antibody (559942, BD Biosciences), PE-conjugated CD24 antibody (555428, BD Biosciences).
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2

CD44v9 Expression and Cell Cycle Analysis

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Cells were detached and incubated with APC-conjugated CD44 antibody (BD-PharMingen, San Diego, CA, USA 559942) or isotype control APC-conjugated IgG2B (BD-PharMingen) in PBS BSA 1% (Sigma) for 30 minutes on ice prior to flow cytometric analysis. Sytox Blue Stain (Life Technologies, Eugene, OR, USA S34857) was added to exclude dead cells. For CD44v8-10 staining 1×105 cells were incubated with 3 μg/ml anti-human CD44v9 primary antibody (clone: RV3) (Cosmo Bio Co. Ltd, Tokyo, Japan LKG-M001) in PBS 0.2 % BSA for 45 minutes at 4°C. Cells were then incubated with the secondary antibody APC-labeled Goat anti-Rat IgG (H+L) (Invitrogen, Carlsbad, CA, USA A10540) for 30 minutes at 4°C. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data analyzed employing FCS5 express Software (De Novo Software). For cell cycle analysis, after labeling for CD44v9, the cells were fixed with 70% ethanol, a few drops at a time mixing the cells, and incubated on ice for 30 minutes. The cells were centrifuged at 500 x g for 10 minutes, washed once in PBS by centrifugation and resuspended in 1 ml PBS containing 5 μg/ml of propidium iodide. Samples were assayed by CyAn ADP flow cytometer and analyzed employing FCS5 express Software.
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3

Quantification of Cancer Stem Cells

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Cells to be analysed were trypsinised, washed and incubated for 1 h in antibody diluted 1:100 in PBS. Flow cytometry was performed on an Accuri Flow Cytometer (BD Biosciences) and analysis of results was performed using a FlowJo software package. APC-conjugated CD44 antibody was purchased from BD Pharmingen, PE-conjugated ALDH1 antibody was purchased from Stratech.
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4

Isolation of CAIX+ and CAIX- Subpopulations

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Flow cytometry was carried out as previously described [28 (link)]. Samples were analyzed using a DakoCytomation Cyan machine, or sorted using a MoFlo cell sorter (Beckman Coulter). The bottom 10% of CAIX negative subpopulation and the top 10% of CAIX positive subpopulation were collected for further experiments. The following primary antibodies were used FITC conjugated CAIX (R&D systems), APC conjugated CD133 (Miltenyi Biotec), APC conjugated CD44 antibody (BD Biosciences) and PE conjugated CD24 antibody (BD Biosciences).
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5

CD44+ Cell Sorting for Cancer Research

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For sorting experiments, PC3 and DU-145 cells were stained with APC-conjugated CD44 antibody (BD-PharMingen, San Diego, CA, USA). Cell populations were analyzed post-sorting to ensure purity of sorting before progressing with additional experiments. Apoptotic cells were excluded by elimination of cells positive for Fixable Viability Stain 780 (FSV780) (BD Biosciences, San Jose, CA, USA). FACS was performed with a FACSAria Cell Sorter (BD Biosciences, San Jose, CA, USA). Three sorting experiments for parental PC3 and DU-145 and two experiments for PC3-GFP were performed.
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