The cell surface antigen profile of paired mCPCs (n = 6 donors) was analyzed by flow cytometry. mCPCs at passage 3 were harvested by trypsin digestion, and antibodies were stained individually (phycoerythrin (PE)-conjugated monoclonal antibodies against human CD29, CD44, CD73, and CD166; fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against human CD45, CD90, and CD271; and allophycocyanin (APC)-conjugated antibodies against CD31 and CD105; eBioscience) for 30 min in the dark at 4 °C. After 2 washes with phosphate-buffered saline (PBS), events were collected by flow cytometry with a FACScalibur system (BectoneDickinson), and the data were analyzed using FlowJo 7.6 software.
Pe conjugated monoclonal antibodies
Manufactured by Thermo Fisher Scientific
PE)-conjugated monoclonal antibodies are fluorescently-labeled immunoglobulin molecules used for the detection and identification of specific target antigens or cells in various research and diagnostic applications. They consist of a monoclonal antibody covalently linked to the fluorescent dye phycoerythrin (PE).
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3 protocols using pe conjugated monoclonal antibodies
1
Immunophenotyping of Mesenchymal Progenitor Cells
2
Cell Surface Antigen Profiling
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Cells were dissociated, washed, and re-suspended in 0.5% BSA, 2 mM EDTA solution in PBS. Cells were stained with PE-conjugated monoclonal antibodies (eBioscience) in solution at 4 °C for 30 min. Rinses were performed with 0.5% BSA, 2 mM EDTA at 4 °C. Alexa Fluor 488 or PE-conjugated isotype-specific IgG were used as control. Flow cytometry was performed using FACSAria II (BD) cell sorter and data was analyzed using the FlowJo software.
3
Quantifying Death Receptor Expression in CRC
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Cell surface expression of pro-apoptotic death receptors in CRC cell lines was analysed incubating 1x10 5 cells with anti-DR4, anti-DR5 or isotype control PEconjugated monoclonal antibodies, (eBioscience) at 4 ºC in PBS containing 5% FCS for Cytotoxicity assays were performed seeding cells (1x10 5 cells) onto cover slips placed in 6-well plates with complete medium, and treated with sTRAIL and LUV-TRAIL at 500 ng/mL for 6 hours. After that, nuclear morphology was analysed by fluorescence microscopy by staining with Hoechst 33342 (Life Technologies) as previously described [39] .
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