Free histones are not detectable in sera of baboons challenged with E coli. (A) Schematic overview of total histone precipitation by Poros HS; nucleosome precipitation by an anti-DNA antibody coupled to sepharose, followed by precipitation of remaining free histones by Poros HS. (B-C) FSAP-deficient serum was spiked with 5 to 20 µg/mL of histones (B) or nucleosomes (C) and incubated with Poros HS, a strong cation exchange resin, to precipitate both histones and nucleosomes. In parallel, similarly spiked samples were incubated with an anti-DNA antibody coupled to sepharose to immunoprecipitate all nucleosomes, but not free histones, before incubation with Poros HS to precipitate the remaining free histones. Samples were separated by SDS-PAGE, and the presence of histone H3 in the precipitated samples was determined using anti–histone H3 antibody on immunoblot. (D) The presence of nucleosomes and free histones was analyzed in serum of 2 baboons that had been lethally challenged with E coli. As before, total histones, nucleosomes, or free histones were separated on gel, and histone H3 was visualized (D) and quantified (E) using a histone titration on the same blot as exemplified for baboon 2. (E) Histone levels as determined with densitometric analysis. (F) Nucleosome levels as determined in the sera by ELISA. (G) FSAP-AP levels were determined by ELISA to verify FSAP activation. Blots are representative of 3 independent experiments, except for the baboon samples, which are representative of 2 independent experiments because of limited sample availability. Arrows indicate intact histone H3 or a cleavage product of histone H3.