Detection of NETs was assessed with cfDNA and CitH3, considered one of the most specific markers of NET formation [58 (link)]. As found in previous studies, NETs can predict more severe episodes of COVID-19 pneumonia and estimate different clinical trajectories [59 (link)].
Plasma samples were mixed with a monoclonal mouse anti-histone biotinylated antibody (Component 1, Cell Death Detection ELISAPLUS) in a streptavidin-coated plate (Component 9). A rabbit polyclonal anti-histone-H3 (citrullinated R17 + R2 + R8) (ab81797; Abcam Inc., Waltham, MA, USA) antibody was used in a second step. Detection was carried out with a peroxidase-linked antibody (GE Biosciences, Barcelona, Spain). A pool of samples from normal subjects was used to normalize values. It was included in all microplates, being expressed as individual absorption values.
To determine cfDNA, plasma was diluted 1:10 with phosphate-buffered saline (PBS (in mmol/L: NaCl 137, KCl 2.7, Na2HPO4 10, KH2PO4, pH 7.4)) and mixed with an equal volume of 1 mm SytoxGreen (Invitrogen, Carlsbad, CA, USA). Fluorescence was determined in a fluorescence microplate reader (Gemini XPS; Molecular Devices, Sunnyvale, CA, USA). A calibration curve was generated with calf thymus DNA (Invitrogen) in PBS. More detailed information is provided elsewhere [60 (link)].
Plasma samples were mixed with a monoclonal mouse anti-histone biotinylated antibody (Component 1, Cell Death Detection ELISAPLUS) in a streptavidin-coated plate (Component 9). A rabbit polyclonal anti-histone-H3 (citrullinated R17 + R2 + R8) (ab81797; Abcam Inc., Waltham, MA, USA) antibody was used in a second step. Detection was carried out with a peroxidase-linked antibody (GE Biosciences, Barcelona, Spain). A pool of samples from normal subjects was used to normalize values. It was included in all microplates, being expressed as individual absorption values.
To determine cfDNA, plasma was diluted 1:10 with phosphate-buffered saline (PBS (in mmol/L: NaCl 137, KCl 2.7, Na2HPO4 10, KH2PO4, pH 7.4)) and mixed with an equal volume of 1 mm SytoxGreen (Invitrogen, Carlsbad, CA, USA). Fluorescence was determined in a fluorescence microplate reader (Gemini XPS; Molecular Devices, Sunnyvale, CA, USA). A calibration curve was generated with calf thymus DNA (Invitrogen) in PBS. More detailed information is provided elsewhere [60 (link)].
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