Flow Cytometry for Stem Cell Markers

The expression of CD133, EpCAM and CD44 surface antigens were evaluated by flow cytometry following a previously described procedure [21 (link)]. In a one-tube assay, cells were stained with phycoerythrin (PE)-conjugated anti-CD133/2 (293C3) and fluorescein isothiocyanate (FITC)-conjugated anti-CD326 (EpCAM) (Miltenyi Biotec, Bologna, I) or with PE-conjugated anti-CD133, FITC-conjugated anti-EpCAM and allophycocyanin (APC)-conjugated anti-CD44 (Becton Dickinson, San José, CA) monoclonal antibodies. All samples were analyzed by a FACSCalibur flow cytometer (Becton Dickinson) with the CellQuest Pro 6.0 software (Becton Dickinson). 20,000 non-debris events in the morphological gate were recorded for each sample. All antibodies were titrated under assay conditions and optimal photomultiplier (PMT) gains were established for each channel, as previously reported [22 (link)].
Data were analysed using FlowJo™ software (TreeStar, Ashland, OR) and reported as percentage of positive cells or as mean fluorescence intensity (MFI) values.