The CD133/2 and CD24 expression was quantified using flow cytometry using a standard protocol. Briefly, cells were labeled with mouse anti-human CD24-Percp-Cy 5.5 (BD Biosciences; San Jose, CA, USA) and mouse anti-human CD133/2-PE (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) antibodies, while mouse Percp-Cy 5.5-IgG1 K isotype antibodies (BD Biosciences; San Jose, CA, USA) and PE-IgG2b isotype (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) served as isotype controls. Then, cells were washed and analyzed on a FACSCaliber flow cytometer (BD Biosciences; San Jose, CA, USA). Apoptosis or necrosis was determined using the AnnexinV-PE/7-AAD Apoptosis Detection Kit (BD Biosciences; San Jose, CA, USA) following the manufacturer’s instructions, and cell cycle was determined using BD CycletestTM Plus DNA Reagent Kit (BD Biosciences; San Jose, CA, USA). All experiments were repeated in triplicate.
Mouse anti human cd133 2 pe
Manufactured by Miltenyi Biotec
Sourced in United States
The Mouse anti-human CD133/2-PE is a fluorochrome-conjugated monoclonal antibody that specifically binds to the human CD133/2 antigen. This antibody can be used for the identification and analysis of CD133/2-positive cells in flow cytometry applications.
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2 protocols using mouse anti human cd133 2 pe
1
Quantifying CD133/2 and CD24 Expression
2
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.
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