Mouse igg1 fitc

Cells were lifted using 1% (w/v) EDTA, centrifuged (800 x g for 2 min) and resuspended in PBS (5×10⁵ cells/ml) containing 0.25 µg of allophycocyanin (APC), phycoerythrin (PE) or FITC directly conjugated antibodies (anti-CD44 APC, anti-CD24 FITC anti-ABCG2 PE and anti-integrin α6 APC (eBioscience, Inc., San Diego, CA, USA), and anti-CD133 PE (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), or used as unstained controls. Isotype controls (mouse IgG2b PE, Rat IgG2b APC, mouse IgG1 FITC, mouse IgG1 PE; eBioscience, Inc.) were used to account for any non-specific antibody binding to live cells by establishing a gating threshold (limit 0.5%) according to fluorescence intensity and calculated compensation. All antibodies were used at a 1:400 dilution (0.25 µg) and samples were incubated at 4°C for 1 h before harvesting of cells for analysis). The FACSAria II flow cytometer was used to record 50,000 events prior to analysis using FlowJo software. The APC fluorophore was excited at 633 nm and emission recorded in the 620/20 filter channel. Both the FITC and the PE fluorophores were excited at 488 nm and emission recorded in the 530/30 or 585/42 filter channels, respectively. Compensation controls were used to establish values for fluorescent overlap.