Surface and intracellular staining were performed using routine protocols as described before [52 (link)]. The following antibodies were used: Ki67-APC (Biolegend, San Diego, CA, USA), CD44-APC and CD326/EpCAM-APCVio779 from Miltenyi Biotec (Bergisch Gladbach, Germany). Apoptosis was evaluated by Annexin V/Iodure Propidium staining using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen™ eBioscience™).
ALDH activity was measured by flow cytometry with ALDEFLUOR (StemCell Technologies, Vancouver, BC, Canada) as recommended by the manufacturer. Briefly, 10 × 105 cells were suspended in ALDEFLUOR assay buffer containing an ALDH substrate (BODIPY-aminoacetaldehyde-diethyl acetate, BAAA-DA) and incubated at 37 °C for 45 min. A negative control for each sample was generated by incubation with the ALDH inhibitor diethylaminobenzaldehyde. Cells were washed with the ALDEFLUOR buffer and analyzed by flow cytometry in a flow cytometer (BD Biosciences, San Jose, CA, USA).
ALDH activity was measured by flow cytometry with ALDEFLUOR (StemCell Technologies, Vancouver, BC, Canada) as recommended by the manufacturer. Briefly, 10 × 105 cells were suspended in ALDEFLUOR assay buffer containing an ALDH substrate (BODIPY-aminoacetaldehyde-diethyl acetate, BAAA-DA) and incubated at 37 °C for 45 min. A negative control for each sample was generated by incubation with the ALDH inhibitor diethylaminobenzaldehyde. Cells were washed with the ALDEFLUOR buffer and analyzed by flow cytometry in a flow cytometer (BD Biosciences, San Jose, CA, USA).
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