Flow Cytometric Analysis of ALDH Activity

Flow cytometry was carried out as previously described [68 (link), 69 (link)]. For ALDH staining, cells from were stained with the Aldefluor reagent (Stem Cell Technologies) for 30 minutes in a 37°C water bath according to the manufacturer’s protocol. ALDH⁺ cells were identified by based on cells stained with Aldefluor in the presence of diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH, used to control for background fluorescence. For intracellular staining, cells were fixed with 2% formalin for 10 minutes, followed by permeabilization with 1% NP40 for 10 minutes at room temperature. Cells were stained with rabbit monoclonal anti-pY397-FAK (Invitrogen), rabbit polyclonal anti-FAK (Cell Signaling), or mouse monoclonal anti-ALDH (BD Biosciences) antibodies followed by anti-rabbit-APC and anti-mouse-FITC antibodies (Invitrogen). Annexin V staining was done according to manufacturer’s protocol (eBioscience).

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Begum A., Ewachiw T., Jung C., Huang A., Norberg K.J., Marchionni L., McMillan R., Penchev V., Rajeshkumar N.V., Maitra A., Wood L., Wang C., Wolfgang C., DeJesus-Acosta A., Laheru D., Shapiro I.M., Padval M., Pachter J.A., Weaver D.T., Rasheed Z.A, & Matsui W. (2017). The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma. PLoS ONE, 12(7), e0180181.

Publication 2017

Aldefluor reagent anti-pY397-FAK anti-FAK Annexin V

Annexin v Anti antibodies Antibodies Bath Cells Fitc Flow cytometry Fluorescence Formalin Intracellular Mouse Rabbit Stem cell

Corresponding Organization : Verastem (United States)

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Variable analysis

independent variables

ALDH staining
Intracellular staining with rabbit monoclonal anti-pY397-FAK, rabbit polyclonal anti-FAK, or mouse monoclonal anti-ALDH antibodies

dependent variables

Identification of ALDH+ cells
Phosphorylation of FAK (pY397-FAK)
Expression of FAK
Expression of ALDH

control variables

Staining with diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH, to control for background fluorescence
Cell fixation with 2% formalin and permeabilization with 1% NP40 for intracellular staining

positive controls

Staining with Aldefluor reagent to identify ALDH+ cells

negative controls

Staining with DEAB to control for background fluorescence in ALDH+ cell identification

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