Guava tunel assay

Cells were treated with MTX(OEt)₂ solution and MTX(OEt)₂-LNC at concentrations of 20, 10, and 5 μM, and also with LNC and LNC plus MTX(OEt)₂ solution at the equivalent amounts for 48 hours and the Guava® TUNEL assay (Merck KGaA) was conducted following the manufacturer’s instructions. Briefly, treated cells were subjected to cell fixation procedure with 50 μL of 4% (w/v) paraformaldehyde in phosphate-buffered saline for 60 minutes at 4°C and then with 200 μL of ice-cold 70% (v/v) ethanol at −20°C for at least 18 hours. For the staining procedure, 1.5×10⁴ to 1.0×10⁵ of fixed cells were washed twice followed by addition of 25 μL of DNA Labeling Mix (Roche, Basel, Switzerland) for 60 minutes at 37°C. At the end of the incubation time, cells were centrifugated and resuspended in 50 μL of the Anti-BrdU Staining Mix (Merck KGaA). Cells were incubated in the dark at room temperature for 30 minutes, and samples were acquired on flow cytometry (Guava® Flow Cytometry easyCyte™ System). In this assay, terminal deoxynucleotidyl transferase (TdT) catalyzes the incorporation of BrdU (5-bromo-2’-deoxyuridine) residues into the fragmenting nuclear DNA of apoptotic cells at the 3′-hydroxyl ends by nicked-end labeling. Tetramethylrhodamine-conjugated anti-BrdU antibody binds to the incorporated BrdU residues, labeling the mid- to late-stage apoptotic cells.