Cell death detection elisa

Analysis of cfDNA was performed using Quant-iT™ PicoGreen^® dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The assay was read on a Varioskan LUX (Thermo Fisher).
Measurement of H3Cit from plasma and preparation of H3Cit standard was performed as described in detail before [24 (link)]. Briefly, a 96 well plate was precoated with anti-histone antibody overnight at 4 °C (Cell death detection ELISA, Sigma Aldrich, St. Louis, MO, USA). After blocking with incubation buffer, standards, controls, and samples were incubated for 1.5 h at room temperature. Following washing with phosphate-buffered saline (PBS) + Tween (0.05%; Sigma Aldrich), an anti-H3Cit antibody (1:1000 ab5103; Abcam, Cambridge, MA, USA) was added and incubated for 1.5 h. After washing, anti-rabbit horseradish peroxidase- (HRP) conjugated antibody was applied for 1 h (1:5000; goat- anti-rabbit IgG HRP; Bio Rad, Laboratories, Hercules, CA, USA). After washing, incubation with TMB (3,3′,5,5′-tetramethylbenzidine; Sigma Aldrich) for 25 min resulted in a colorimetric change. The reaction was stopped using 2% sulfuric acid. The plate was read at 450 nM at on a Varioskan LUX (Thermo Fisher).

An in-house ELISA was used to quantify MPO-DNA complexes. Briefly, after overnight coating with anti-MPO antibody (2 μg/mL; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% BSA in PBS for 2 hours at room temperature. The plate was subsequently washed before incubation for 90 minutes at room temperature with 20% human or mouse plasma in blocking buffer. The plate was washed 5 times, and then incubated for 90 minutes at room temperature with anti-DNA antibody (1:10; Cell Death Detection ELISA, 11544675001, Sigma-Aldrich). After 5 washes, the plate was developed with ABTS substrate (10102946001, Sigma-Aldrich).

HaCaT or HPK seeding and experimental conditions were as for luciferase assay. Briefly, cells were lysed and apoptosis measured by a commercial kit revealing presence of apoptotic cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISA, SigmaAldrich, Milan, Italy).