Aldefluor reagent

THP-1 cells were incubated for 30 min with medium containing DMSO or inhibitors at the concentration of 33 μM then washed and incubated with ALDEFLUOR reagent (STEMCELL Technologies) in assay buffer according to the manufacturer’s instruction. Fluorescence derived from substrate metabolized by cellular ALDH were measured by Gallios cytometer.

Flow cytometry analysis or flow cytometry cell sorting was conducted using PE-conjugated mouse anti-human CD133 (Miltenyi Biotec) or its respective isotype control. ALDEFLUOR reagent (Stem Cell Technologies) was used for the immunofluorescent detection of intracellular ALDH enzyme activity. Samples were analyzed on BD FACSCanto II (BD Biosciences) with data analyzed by FlowJo (Tree Star Inc.).

Cells were stained with Aldefluor reagent as per manufacturer's instructions (Stemcell Technologies, Cat# 01700) and the percentage of cells with high Aldehyde Dehydrogenase (ALDH) activity were analyzed. Human breast cancer stem cell marker staining was determined by labeling human cells with CD44-APC and CD24-PE antibodies (BD Biosciences, Cat# 559942 and 555428). Dissociated mammary tumor cells and murine cell lines were stained with CD24-PE, CD29-FITC, CD31-APC, TER-119-APC (BD Biosciences, Cat# 553262, 561796, 561814, and 561033) and CD45-APC (Biolegend, Cat# 103111) antibodies, analyzed using the FACS Diva software, and sorted using the Aria Illu sorter (BD Biosciences) [47 (link)].

For assessing the proportion of stem cells we have used ALDEFLUOR reagent (STEMCELL Technologies), which has been shown to be specific for stem/progenitor cells in mammary epithelial cell populations. A previous study [62 (link)] using a derivative of one of our strains (184A1), revealed that pre-sorting cells using the ALDEFLUOR kit improves mammosphere generation by ~3 fold. Radiated and sham exposed cell strains were grown in 100 mm dishes and trypsinized 9 days following radiation exposure. Single cells in suspension were filtered using a 35 μ nylon mesh, counted using a hemocytometer and set up at the suggested concentrations for ALDEFLUOR labeling as per established protocols (STEMCELL technologies Inc., Vancouver). DAPI was included in the staining protocol for exclusion of dead cells that stain positive for DAPI (DAPI+). Cells expressing high levels of aldehyde dehydrogenase activity (ALDH+) were identified by their bright green fluorescence and enumerated from the total mammary cell population on a BD FACS Vantage SE flow sorter based on the FL1 channel. Gates for the ALDH+ population were defined using the supplied negative control that includes DEAB, an inhibitor of ALDH activity. Data are presented as proportion of stem cells in the radiated samples relative to sham-irradiated controls.

Mammospheres were obtained by culturing the dissociated MCF-7 cells for 2 weeks in SCM, as described in Section 4.2 (Sphere Formation Assay). For measuring ALDH-activity in mammospheres, spheres were gently trypsinized and pipetted to obtain single cell suspension. MCF-cells cultured in the presence of antibiotics and mammosphere-derived cells were stained with the ALDEFLUOR reagent (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturers protocol. Briefly, the total number of 1 × 10⁶ cells were re-suspended in 1 mL assay buffer; further 6 μL of ALDEFLUOR reagent was added, and 500 µL of this cell suspension was transferred into a new tube with 6 μL of diethylaminobenzaldehyde (DEAB) regent. Cells were incubated for 30 min at 37 °C, centrifuged, and resuspended in 500 μL of Assay Buffer and subjected to an analysis on a FACSAria III flow cytometer (Becton Dickinson, NJ, USA).

Fluorescence-activated cell sorting (FACS) was used to isolate the ALDH and CD44 double-positive/double-negative subpopulation. Briefly, cells were suspended and incubated in ALDEFLUOR™ Assay Buffer containing ALDEFLUOR™ reagent (StemCell Technologies, Canada) at 37°C for 30 min and centrifuged for 10 min at 300 g; the supernatant was then discarded. APC-conjugated CD44 antibody (Miltenyi Biotec, Germany) was added to the cells resuspended in Flow Buffer (Miltenyi Biotec, Germany) with the FcR Blocking Reagent. Then the mixture was incubated in the dark for 30 min at 4°C. Cells were stained with the ALDH inhibitor diethylaminobenzaldehyde (DEAB), and anti-IgG-APC was used as a negative control for flow cytometry gating.