Fluorescent counting beads

Citrated blood freshly collected from HIT patients and healthy donors was diluted in PBS and stained with anti-CD15 (Alexa 647, BD 562369), anti-CD16 (APC-Cy7, BD 557758), anti-CD41a (V450, BD 561425), anti-citrullinated histone H3 (ab5103), anti-MPO (PE, BD 341642) and Affinipure donkey anti-rabbit (Alexa 488, Jackson ImmunoResearch Laboratories 711545152) antibodies. Samples were fixed with paraformaldehyde and analysed by flow cytometry (CantoII, BD Bioscience). Citrated blood from healthy donors was also treated in vitro with HIT or control antibodies, labelled and analysed as described above. Fluorescent counting beads (Invitrogen C36950) were used to derive absolute cell numbers by flow cytometry following the manufacturer’s instructions. Platelet counts from animal experiments were determined by the number of events acquired in 60 s. LDG and monocytes from human and mouse experiments isolated using Histopaque separation media (Sigma)⁴⁹ were identified using anti-CD15 (Alexa 647, BD 562369), anti-CD16 (APC-Cy7, BD 557758, anti-CD14 (V500, BD 561391), anti-Ly6G (V450, BD560603) and anti-CD11b (APC, BioLegend 101212).

Lavage fluid cell suspensions (200 μL) were incubated for 20 min with rat anti-mouse CD16/CD32 to block nonspecific Fc binding then stained with anti-mouse Ly-6G (1A8) for 30 min. Brain cell suspensions were prepared by enzymatic digestion and density gradient centrifugation and cells incubated with anti-CD16/CD32 before staining with anti-mouse Ly6G-Pacific Blue. Absolute cell counts were determined by the addition of 50 μL fluorescent counting beads (Invitrogen, Paisley, UK). Flow cytometry was performed on a CyAn™ ADP Flow Cytometer (Dako UK Ltd, Ely, UK) or BD Fortessa (BD Biosciences USA) and data was analyzed using Summit 4.0 software.

Lavage fluid (200 μl) or blood (50 μl blood + 50 μl buffer: 0·1% bovine serum albumin, 1 mm EDTA in PBS) samples were incubated for 20 min with 1 : 200 rat anti‐mouse CD16/CD32 to block non‐specific Fc binding. Cocktails of fluorophore‐conjugated antibodies were added for 30 min, to detect Ly‐6G, Ly‐6C, CD45, B220, CD3, MHC‐2, Gr‐1, CD11b, CD115, CD41 and CD61. Red blood cells in samples were lysed by the addition of 450 ml FACS Lysing Solution (BD Biosciences, Oxford, UK). Absolute numbers of cells were determined through the use of TruCOUNT™ tubes (BD Biosciences), or by the addition of 50 μl fluorescent counting beads (Invitrogen, Paisley, UK). Flow cytometry was performed on a CyAn™ ADP Flow Cytometer (Dako UK Ltd, Ely, UK) equipped with 405‐nm, 488‐nm and 633‐nm lasers using summit 4.0 software (Dako UK Ltd, Ely, UK). Cell populations were determined on summit 4.0 software via positive labelling of relevant markers. For blood samples, a minimum of 1000 beads, 1000 neutrophils or 5000 leucocytes (whichever threshold occurred last) were acquired per sample. For lavage samples, a minimum of 20 000 cellular events were acquired per sample.