4 laser attune nxt acoustic cytometer

The AldeRed with 588-A ALDH Detection Assay (SCR150, Millipore) was used, following the manufacturer's instructions. Briefly, 2×10⁵ cells were resuspended in AldeRed assay buffer containing the AldeRed 588-A substrate. The cell suspension was split into two fractions, one half of which served as a control, and was transferred to a new tube containing the specific ALDH1 inhibitor diethylamino-benzaldehyde (DEAB). Cells were then incubated for 45 min at 37°C in complete darkness. Next, cells were centrifuged (300 g, 5 min), their supernatants discarded, and cell pellets resuspended in 500 μL of cold AldeRed assay buffer. Samples were stored on ice and darkness prior to flow cytometry analysis (4-laser Attune NxT Acoustic Cytometer; Thermo Fisher Scientific). DAPI was used to mark and exclude dead cells, and data was analyzed using FlowJo v9.3 software (Tree Star Inc., Ashland, OR).

Cells were plated at 5 × 10⁵ cells/well in a 6-well plate, transfected with the aptamers during 24 h, resuspended, and stained with Annexin V Apoptosis detection kit (Canvax, Córdoba, Spain) as described previously [37 (link)] and analyzed with a 4-laser Attune NxT Acoustic Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Results were analyzed using FCSalyzer 0.9.22 alpha Software.

SA-β-Gal activity was measured following two different approaches. (i) AGS, MKN45 and PANC1 cells (5 × 10⁴) were seeded in 24-multiwell dishes and incubated overnight. Then, cells were treated for 24 h with the IC₅₀ concentration of each compound. For flow cytometry cell senescence detection, cells were tripsinized, fixed for 15 min with PFA 4% and stained with the CellEvent Senescense Green Flow Cytometry Assay Kit (C10840, Invitrogen) according to manufacturer’s instructions. CellEvent Senescense Green Probe was used at a 1:500 dilution and fluorescence was detected using the filter (Ex488nm/Em530/30) BL1 with a 4-laser Attune NxT Acoustic Cytometer (Thermo Fisher Scientific). (ii) For histochemical staining assays, AGS cells (2 × 10⁵) were seeded in a 6-well plate, incubated overnight, and treated with CDDP (20 µM), H₂O₂ (200 µM) and a IC₂₅ concentration of I5 (15 µM) or I6 (15 µM). After 3 h, the treatments were removed, new supplemented medium was added, and cells were incubated for an additional 24 h. Finally, SA-β-Gal staining was performed using the Senescence Cells Histochemical Staining Kit (CS0030-1KT) according to the manufacturer’s instructions.