Anti icam 1

In order to quantify NMV internalisation, HCAEC (5 × 10⁴) were seeded onto 24-well tissue culture plates and cultured overnight. The next day HCAEC were incubated with media alone, media + TNF (1 ng mL⁻¹; R&D Systems, Abingdon, UK), media + anti-ICAM-1 (100 ng mL⁻¹; Biolegend, London, UK, catalogue number: 322704) or media + TNF + anti-ICAM-1 for 4 h. Consequently fluorescently labelled NMVs (0.4 × 10³ µL⁻¹) were added and cells incubated for 2 h at 4 °C, room temperature or 37 °C (as specified in the corresponding figure legends). The use of low temperatures to inhibit endocytosis has been used by others to demonstrate internalisation is a metabolically active process i.e. endocytosis dependent^{59 (link),60 (link)}. Cells were then washed to remove excess NMVs and detached using a trypsin/EDTA solution. Cells were washed and, immediately prior to flow cytometry analysis, Trypan blue (1 mg mL⁻¹) was added to each sample in order quench fluorescence of residual surface bound NMVs^{61 (link),62 (link)}, thus ensuring any fluorescent signal detected was from internalised NMVs only. Data were analysed for mean fluorescence intensity using an LSRII flow cytometer using FACSDiva acquisition software.

Human PBMCs were seeded in 96-well plates and pre-treated with 1 and 10 μM of KRO-105714. After incubating for 30 min, these cells were treated with 10 μM SPC and/or 5 μg/ml PHA. PBMCs were analyzed by flow cytometry using 1 μg/ml of anti-CD45-APC (BioLegend, San Diego, CA, USA), anti-ICAM-1 (BioLegend) and anti-CD44 antibodies in phosphate-buffered saline (PBS) plus 1% fetal bovine serum (FBS) on ice for 10 min. Cells were then washed and analysed. Staining data were collected using a FACSCanto II Cytometer (BD Biosciences). To set the gates for defining positive and negative cells at 5000 cells in multicolor staining, samples were stained with a mixture of all antibodies [50 (link)].