Human anti ec7 mab 3677

Human U2OS cells expressing variant PCDH1 were seeded in 6-well plates 24 h prior to immunostaining. Cells were chilled on ice for 10 min (min) and blocked with chilled PBS/10% FBS for 30 min at 4 °C. Surface PCDH1 was stained using human anti-EC7 mAb-3677 (5 µg/mL, Donnelly Centre and Department of Molecular Genetics, University of Toronto) followed by anti-human IgG Alexa Fluor^TM 488 pAb (1:500 dilution, catalog No. A-11013, Thermo Fisher) for 1 h at 4 °C. After washing, cells were resuspended in PBS/2% FBS, and those intended to be sorted were stained with TO-PRO^TM3 Ready Flow^TM Reagent (Invitrogen) to identify and exclude dead cells. Cells were passed through a 0.41 µm Nylon Net Filter (Millipore) and analyzed using an LSRII Flow Cytometer (BD Biosciences, CellQuest Pro software, V.6.1) and FloJo V.10 software. Subpopulations of cells expressing PCDH1(10a.a.) were isolated by FACS (NanoCellect WOLF Cell Sorter, WOLFViewer software, V.2.4) and verified following the staining method described above.

Human U2OS cells expressing variant PCDH1 were seeded in 6-well plates 24 h prior to immunostaining. Cells were chilled on ice for 10 min (min) and blocked with chilled PBS/10% FBS for 30 min at 4 °C. Surface PCDH1 was stained using human anti-EC7 mAb-3677 (5 µg/mL, Donnelly Centre and Department of Molecular Genetics, University of Toronto) followed by anti-human IgG Alexa Fluor^TM 488 pAb (1:500 dilution, catalog No. A-11013, Thermo Fisher) for 1 h at 4 °C. After washing, cells were resuspended in PBS/2% FBS, and those intended to be sorted were stained with TO-PRO^TM3 Ready Flow^TM Reagent (Invitrogen) to identify and exclude dead cells. Cells were passed through a 0.41 µm Nylon Net Filter (Millipore) and analyzed using an LSRII Flow Cytometer (BD Biosciences, CellQuest Pro software, V.6.1) and FloJo V.10 software. Subpopulations of cells expressing PCDH1(10a.a.) were isolated by FACS (NanoCellect WOLF Cell Sorter, WOLFViewer software, V.2.4) and verified following the staining method described above.

Human U2OS cells expressing variant PCDH1 were seeded in 6-well plates 24 h prior to immunostaining. Cells were chilled on ice for 10 min (min) and blocked with chilled PBS/10% FBS for 30 min at 4 °C. Surface PCDH1 was stained using human anti-EC7 mAb-3677 (5 µg/mL, Donnelly Centre and Department of Molecular Genetics, University of Toronto) followed by anti-human IgG Alexa Fluor^TM 488 pAb (1:500 dilution, catalog No. A-11013, Thermo Fisher) for 1 h at 4 °C. After washing, cells were resuspended in PBS/2% FBS, and those intended to be sorted were stained with TO-PRO^TM3 Ready Flow^TM Reagent (Invitrogen) to identify and exclude dead cells. Cells were passed through a 0.41 µm Nylon Net Filter (Millipore) and analyzed using an LSRII Flow Cytometer (BD Biosciences, CellQuest Pro software, V.6.1) and FloJo V.10 software. Subpopulations of cells expressing PCDH1(10a.a.) were isolated by FACS (NanoCellect WOLF Cell Sorter, WOLFViewer software, V.2.4) and verified following the staining method described above.