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Aldehyde dehydrogenase (ALDH) flow cytometry assay

SV Siru Virtanen
RS Reiner Schulte
JS John Stingl
CC Carlos Caldas
MS Mona Shehata
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Human breast single cell suspensions were treated to detect the enzyme activity of aldehyde dehydrogenase (ALDH) using the Aldefluor Kit (StemCell Technologies) as per the manufacturer’s instructions. The cells were then preblocked with 10% normal rat serum (Sigma) and stained with the following antibodies: CD31-APC/Cy7 (Clone WM-59), CD45-APC/Cy7 (Clone HI30), EpCAM-PE, CD49f-PE/Cy7 (Clone GoH3) (all from BioLegend) in combination with one of the following antibodies CD140b-AF647 (Clone 28D4), CD142-AF647 (Clone HTF-1), CD26-AF647 (Clone M-A261), CD34-AF647 (Clone 581), CD340 (Her2)-AF647 (Clone Neu24.7), CD39-AF647 (Clone TU66), CD44-AF647 (Clone G44-26), CD49c-AF647 (Clone C3 II.1), CD66 (a,c,d,e)-AF647 (Clone B1.1/CD66), CD54-AF647 (Clone LB-2), CD55-AF647 (Clone IA10), CD13-AF647 (Clone WM15), CD73-AF647 (Clone AD2), CD15s-AF647 (Clone CSLEX1), CD151-AF647 (Clone 14A2.H1), CD166-AF647 (Clone 3A6), CD282-AF647 (Clone 11G7), CD63-AF647 (Clone H5C6), CD75-AF647 (Clone LN1), SSEA-4-AF647 (Clone MC813-70), TRA-1-81-AF647 (Clone TRA-1-81), CLA-Biotin-AF647 (Clone HECA-452), CD15-AF647 (Clone HI98) (all from BD Biosciences). Cells were then filtered through a 30-μm cell strainer and incubated with DAPI. Human cells were separated using an Influx cell sorter (Becton Dickinson). Single-stained control cells were used to perform compensation manually. Gates were set in reference to fluorescence-minus-one controls. The ALDH+ gate was set in reference to control populations incubated with the ALDH inhibitor DEAB in addition to Aldefluor. Flow cytometry data were analysed using FlowJo™ software.

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