Alexa Fluor® 488 anti-human CD15 (SSEA-1) Antibody

Protocol tips

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	neurospheres were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies) and prepared for fluorescence activated cell sorting (FACS) analysis as we have previously described.	Cells were analyzed using an LSR II Flow Cytometer or FACSCalibur (Becton Dickinson Biosciences) by the UAB Flow Cytometry Core Facility, and the results were expressed as a percentage of gated cells based on antibody binding using FlowJo version 10.0.6 software (Tree Star).

Protocol tips
neurospheres were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies) and prepared for fluorescence activated cell sorting (FACS) analysis as we have previously described.
Downstream tips
Cells were analyzed using an LSR II Flow Cytometer or FACSCalibur (Becton Dickinson Biosciences) by the UAB Flow Cytometry Core Facility, and the results were expressed as a percentage of gated cells based on antibody binding using FlowJo version 10.0.6 software (Tree Star).

Publication protocol

To determine expression of CD133, CD15, and C111, neurospheres were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies) and prepared for fluorescence activated cell sorting (FACS) analysis as we have previously described.25,26 The following fluorochrome-conjugated monoclonal antibodies were used: allophycocyanin-CD133 (Miltenyi Biotec), Alexa Fluor 488–CD15 (BioLegend), and phycoerythrin-CD111 (proline-rich protein 1, nectin-1; BioLegend). Cells were analyzed using an LSR II Flow Cytometer or FACSCalibur (Becton Dickinson Biosciences) by the UAB Flow Cytometry Core Facility, and the results were expressed as a percentage of gated cells based on antibody binding using FlowJo version 10.0.6 software (Tree Star). Mean values from multiple determinations on separate dates and with separate cultures were calculated and paired Student's t-tests were used to determine significance.

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