Cytotoxicity Assay of Histones and Polysialic Acid

5B8 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Gibco, Darmstadt, Germany), including 10% (v/v) fetal calf serum (FCS; Thermo Fisher Scientific, Dreieich, Germany) and 1% Streptavidin at 37 °C and 5% CO₂. 30,000 cells were seeded per well on 96-well plate in 100 µL of RPMI medium, including histones or polySia, and were incubated for 1 h 40 min at 37 °C and 5% CO₂ [32 (link),33 (link)]. Here, the concentration of histones was 60 µg/mL, and the concentration of Neu5Ac or fractionated sialic acid polymers was 40 µg/mL. To determine the cytotoxicity a lactate dehydrogenase (LDH) cytotoxicity assay (BioVision, Milpitas, CA, USA) was applied.