Aldered aldh detection assay

Aldefluor assay was performed using the AldeRed ALDH Detection Assay (Merck) according to manufacturer’s instruction. Flow cytometry was performed using BD ACCURI C6 flow cytometer (BD Biosciences, NJ, USA)

The assay was carried out using AldeRed ALDH Detection Assay (Sigma-Aldrich SCR150). The cells were processed as mentioned in the assay kit.

To determine the intracellular aldehyde dehydrogenase (ALDH) concentration, an equal number of HCC cells were seeded onto six-well dishes. The attached cells were trypsinized and washed with PBS. Cells were stained with AldeRed™ ALDH Detection Assay (Sigma-Aldrich SCR150) for analyzing the ALDH level. Stained cells were analyzed using the FACSCanto II Analyzer (BD Biosciences). The results were performed with FlowJo software.

9 days after culture in Matrigel: Collagen, cells were subject to Aldered ALDH detection assay (Millipore, cat. no. SCR150). Aldered-588 reagent was prepared in assay buffer containing Verapamil to prevent efflux, according to manufacturer's instructions. 5 µl of Aldered solution was added to each with or without DEAB for 60 min at 37°C. Cells were counterstained with DAPI. Fpur images per field at 10× magnification were captured using the FL Auto EVOS Imaging system. Expression was quantified by ImageJ, using approaches described previously (Cheng et al., 2008 (link)). Expression was normalized to DAPI.

The expression of aldehyde dehydrogenase (ALDH) was measured using the AldeRed ALDH Detection Assay (SCR150,Millipore) following the manufacturer’s protocol. TNBC cells were seeded on topographical surfaces attached to 24-well plates at a density of 52,000 cells/well and allowed to attach overnight. The culture medium was then changed to a reduced-serum formulation containing 2% FBS, and cells were cultured for 5 days. Briefly, the cells were washed once with PBS and detached from each well plate using 0.5% trypsin EDTA. The cells were then resuspended in cell culture media (10% FBS) and the cell number was adjusted to 2 × 10⁵ cells per sample. Cells were centrifuged and resuspended in AldeRed assay buffer, and the AldeRed substrate was added to each sample. The samples were incubated at 37°C for 45 min, resuspended in ice-cold AldeRed assay buffer, and maintained on ice during analysis. The cells were analyzed using flow cytometry (BD Accuri™ C6 Plus). Diethylaminobenzaldehyde (DEAB), an ALDH inhibitor, was used as a negative control to gate the ALDH + population.