Cytotoxicity Assay for Histones and PLY

Cells (5 × 10⁴) were seeded in 96-well culture plates and cultured for 24 h in 5% CO₂ at 37 °C until confluent. Histones (0–300 µg/mL, Sigma-Aldrich, Cambridge, UK) or PLY (0–3 µg/mL, produced in house) [20 (link)] were used to treat cells for one hour. The culture medium was collected and immediately centrifuged 200× g, 5 min. Cellular supernatants were stored at −80 °C for lactate dehydrogenase (LDH, Sigma-Aldrich, Cambridge, UK), alanine aminotransferase (ALT, Colorimetric, Abcam, Cambridge, UK), and cardiac troponin I (cTnI, RayBiotech, Peachtree Corners, GA, USA) quantification, as per the manufacturer’s instructions. Adherent cells were immediately washed twice with PBS and cultured in medium containing WTS-8 for 2 h and measured the absorbance (460 nm) for determining cell viability, as described previously [20 (link)].