Slide vi0.4 chambers

Clots were formed from 30% plasma, 0.25 µM Alexa Fluor 488 (AF488) fibrinogen (ThermoFisher Scientific, Massachusetts, USA) and 16 µM phospholipids (Rossix, Molndal, Sweden). In some cases, an AF555 labelled monoclonal antibody against cross-linked fibrin (Zedira, Darmstadt, Germany) was incorporated. Clotting was initiated with 0.1 U/ml thrombin (Sigma-Aldrich, St Louis, USA) and 10.6 mM CaCl₂ before adding to Ibidi µ-slide VI^0.4 chambers (Ibidi GmbH, Gräfelfing, Germany). Patients from the cryo and Fg-C cohort were matched on their fibrinogen concentration in the pre-transfusion sample, so that cohorts were comparable. Representative images are shown in the manuscript. Images were recorded on Zeiss 880 laser scanning confocal microscope with a 63 X 1.40 oil immersion objective using Zeiss Zen 2012 SP1 software (black edition). Images were analysed using FIJI v1.51 and Diameter J plug in.

Cells were plated in ibidi µ-slide VI^0.4 chambers (ibidi GmbH, Martinsried, Germany) and grown to approximately 70–80% confluency, then stimulated with TNF-α and IL-1β for 24 h. After stimulation, cells were washed twice with PBS, blocked for 30–40 min in PBS with 5% goat serum, and then incubated with 2 × 10⁶ antibody-conjugated-MBs per 1 × 10⁵ cells for 1 h with the ibidi slides turned up-side down, allowing MB-cell interaction. Non-bound MBs were washed away and the cell nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Merck, Darmstadt, Germany), then washed and fixed in 4% PFA. A total of six Z-stack images, with a slice thickness of 1 µm, were taken per ibidi channel with a Leica TCS SP5 system (Leica Microsystems, Wetzlar, Germany) confocal microscope using a 60× oil immersion objective. Experimental set-up was repeated minimum 3 times for analysis.