Quantitative Measurement of Citrullinated Histone H3

Citrullinated histone H3 (citH3) was measured as described^{24 (link)}, with minor modifications. Streptavidin-coated plates were incubated with anti-histone biotin (Roche) for 120 minutes. Plates were washed and incubated with plasma samples or standard (citH3, Cayman) for 90 minutes. After washing, anti-histone H3 (1:2000 in PBS + 1% bovine serum albumin [BSA], Abcam) was added for 60 minutes. Plates were washed and incubated with goat anti-rabbit HRP conjugate antibody (1:5000 in PBS + 1% BSA, BioRad) for 60 minutes, washed, and developed with BM Blue POD Substrate (Roche) for 20 minutes. The reaction was stopped by addition of 2 M H₂SO₄ and optical density was measured on a Promega GloMax Discover microplate reader (450 nm, reference 620 nm).
Double-stranded deoxyribonucleic acid (dsDNA) was quantified using the Quant-iT PicoGreen dsDNA Assay (Invitrogen) according to manufacturer´s instructions. Fluorescence was measured on a GloMax Discover microplate reader (Promega).