2.9. ALDEFLUOR Assay

To analyze the aldehyde dehydrogenase (ALDH) activity, an ALDEFLUOR™ kit (Stem Cell Technologies, Durham, NC, USA) was used following the manufacturer indications. Cells were detached from the culture plastic (2D samples) and PCL scaffolds (3D) as explained in Section 2.8, washed with PBS and subsequently resuspended in ALDEFLUOR™ assay buffer at a concentration of 400,000 cells/mL. ALDEFLUOR™ Reagent (BODIPY-aminoacetaldehyde; BAAA) was added to each cell suspension. In order for the background fluorescence to be considered, a negative control for every sample was set by adding ALDEFLUOR™ diethylaminobenzaldehyde (DEAB), an ALDH inhibitor, to each cell suspension prior to adding BAAA in ALDEFLUOR assay buffer. All samples were incubated for 45 min at 37 °C in the dark.

Incubated samples were analyzed with a Cell Lab Quanta flow cytometer (Beckman Coulter Inc., Miami, FL, USA) to quantify the ALDH-positive cell population. The argon ion laser (488 nm) was used as a light source set at a power of 22 mW. Green fluorescence was detected with fluorescent channel 1 (FL1) optical filter (dichroic/splitter, dichroic long-pass: 550 nm, band-pass filter: 525 nm, detection width 505 to 545 nm). Information of a minimum of 10,000 events was recorded in List-mode Data files (LMD) and analyzed using FlowJo 10.2 software (FlowJo LLC, Ashland, OR, USA). Data were not compensated.

First, side-scatter (SS) and electronic volume (EV; equivalent to forward scatter) dot plots were performed and only single cells were selected, excluding debris and cells aggregates (less than 5%). Then, SS and log FL1 dot plots from DEAB samples were created to establish background fluorescence. The ALDH-positive cells’ gate was traced, delimiting the rightmost area and including only the 0.5% of total cell population. BAAA samples were equally processed and ALDH-positive cells gates of respective controls were used to discern the sample percentage of cells with high ALDH activity.