Anti s100a9 antibody

Spheroid cells were lysed, and used to measure reduced (GSH) and oxidized (GSSG) forms of glutathione (GSH/GSSG-Glo assay, Promega). Subsequently, GSH/GSSG ratio was measured according to the manufacuturer's instructions.
Proximity ligation assay (PLA) Proximity ligation assays were performed using Duolink In Situ-Fluorescence (Sigma) according to the manufacturer's instructions. The Accutase-dissociated spheroid cells or transfected HCT116 cells were attached to fibronectin/poly-D-lysine-coated slides and fixed in 4% paraformaldehyde in PBS for 15 min. The cells were then permeabilized in PBS containing 0.3% Triton X-100, blocked with 5% goat serum, and used for immunostaining with the primary antibodies, followed by incubation with Duolink PLA probes and signal amplification. dimedone-mediated PLA was performed as previously described with some modifications (Tsutsumi et al., 2017) . Briefly, the dissociated cells were fixed with 4% paraformaldehyde in the presence of 5 mM dimedone (Wako) for 5 min at room temperature. The cells were washed three times with PBS, permeabilized with 0.1% Triton X-100 and 0.2% BSA in PBS for 10 min, and blocked in PBS containing 1% BSA for 30 min. After incubation with an anti-S100A9 antibody (Santa Cruz Biotechnology) and an anti-cysteine sulfenic acid antibody (Millipore), the cells were subjected to PLA as described above.