Anti human hmgb1 antibody

Primary GECs were cultured in 4-well culture plates (Nunc) containing round glass inserts. Cells were infected at a confluence of 75-80% with either wild-type P. gingivalis or the ndk-deficient mutant strain at an MOI of 100. Six hours later, cells were treated with ATP at a final concentration of 3 mM for an additional 3 h. Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized using 0.01% Triton X-100 solution in PBS and incubated with the primary antibodies. IL-1β was detected using an FITC-conjugated mouse monoclonal antibody (R&D Systems). HMGB1 was detected using a rabbit polyclonal anti-human HMGB1 antibody (Cell Signaling). P. gingivalis infection was detected using anti-P. gingivalis rabbit polyclonal antibody, coupled with a secondary goat-anti-rabbit polyclonal antibody (Invitrogen), and all inserts were mounted onto glass slides using VectaShield mounting medium (Fisher) containing DAPI stain. The intensity of IL-1β and HMGB1 staining was measured using NIH-ImageJ, and measurements of at least 25 cells, originating from at least three separate replicate experiments, were averaged.