Filtermax 3

Since NETs contain both CitH3, MPO and DNA, a CitH3‐DNA or MPO‐DNA complex ELISA was used to detect and quantify soluble NETs in human and mouse serum as described in our previous study.
³¹ (link) Briefly, 96‐well plates were coated with anti‐CitH3 (ab5103, Abcam) or anti‐MPO (ab272101, Abcam) antibodies overnight at 4°C. The plates were then blocked with 2% BSA at room temperature for 2 h. After washing three times, the sample was added to the wells with incubation buffer containing a peroxidase‐labelled anti‐DNA mAb (Cell Death ELISA^PLUS, 11774425001, Roche, Switzerland). After incubation for 2 h, the plates were washed three times. Peroxidase substrate (ABTS) was added, and absorbance was measured by a microplate reader (FilterMax 3, Molecular Devices) at 405 nm. Values for soluble NET formation are expressed as the percentage increase in absorbance above the control.

Cells were seeded at 2.10⁴ cell/100 μl of respective growth medium in a 96 well plate. Polyclonal goat anti-human apoA-1 IgG or polyclonal goat control IgG were added at 150 ug/ml. 1 μM of staurosporine was used as positive control for death induction. Conditions were performed in duplicate. Cell viability was quantified over 4 days using the Cell Titer 96^® AQ_ueous One Solution cell Proliferation Assay (Promega Corp., Madison, WI). Every 24 h, 20 μl of reagent were added to the 100 μl of cell supernatants and incubated at 37°C, 5% CO₂. Absorbance was red after 30 min and 1 h of incubation, at 490nm using a FilterMax3 (Molecular Devices, LLC San Jose, CA, USA).