Af647

Murine samples. Harvested humerus samples were fixed o/n in 10% neutral buffered formalin and then decalcified with 17% EDTA (Osteosoft, Millipore) during 7 days. Samples were processed, paraffin embedded and sectioned (5 μm) for histological studies. Hematoxylin/eosin was performed first to assess quality of the sections of BM samples and visualize vessel integrity. For immunofluorescence (IF) studies heat antigen retrieval was performed in all cases. Primary antibodies are listed in the key resources table. Goat anti-mouse/rabbit/rat secondaries antibodies coupled to AF-488, AF555 and AF647 were used (all from Invitrogen). DAPI was included in the mounting media to label the nuclei. Images were obtained with a Zeiss LSM710 upright confocal microscope. At least 3 images were taken per sample group. To quantify micro-vascular density (MVD), the number of vessel sprouts/mm2 was counted (Bitplane), and data are show as fold over ctrl.

PBMCs were stained with anti-CD4 (#317450 and #300508, Biolegend), anti-CD8 (#344750 and #344714, Biolegend), anti-CD14 (#301815, Biolegend), anti-CD19 (#332224, Biolegend), anti-CD69 (#310904, Biolegend), anti-SIRPG (#336606, Biolegend), anti-CD96 (#338405, Biolegend), AF647- (#A20186, Invitrogen, Carlsbad, CA) conjugated adalimumab and control IgG1 (#403502, Biolegend). Stained cells were collected with FACSCanto or LSRFortessa (Becton Dickenson, Franklin Lakes, NJ), and the data was analyzed with FlowJo software (Becton Dickinson)

Fluorescence signals with broad spectral features were acquired by capturing images of a 30 μL solution of 1 mg/mL of the fluorescent dye AF647 (Invitrogen, USA) dissolved in phosphate buffered saline (PBS) in a well plate (μ-Slide 18 Well—Flat, ibidi GmbH, Germany) using illumination from a 630 nm LED (UHP-T-LED-630, Prizmatix, Israel) and a long pass emission filter (ET700/75 m, Chroma, USA). The mean pixel intensity, S, was calculated in a region of interest (ROI) drawn manually inside the well on the image. For multispectral imaging, the average pixel intensity was extracted from those bands that overlap with the emission spectrum of AF647 (narrow bands: 665 nm, 714 nm, FWHM, 27 nm, 26 nm; broadband: 500–850 nm).
The average pixel intensity, S, was used to determine the signal score S_signal $$S_{signal}=\frac{S-S_{min}}{S_{max}-S_{min}}$$ where S_max and S_min are the maximum and minimum signals calculated across all correction methods, such that scores of 1 and 0 represent the best and worst signal achieved respectively.

Neutrophils were isolated using the EasySep Neutrophil Isolation Kit (Stemcell Technologies), counted, and resuspended in RPMI containing L-glutamine and 5% FCS. Neutrophils were exposed to plasma (10% final concentration) derived from healthy donors (n = 4) or patients with COVID-19 (n = 7) and subsequently stained for indicated activation markers. For selected subgroups, human IL-8 (MilliporeSigma, I1654), anti-human IL-8 antibody (MilliporeSigma, I2519), or reparixin (20 μM, SelleckChem, S8640) were added to either plasma or neutrophils 20 minutes before neutrophil exposure to plasma. Cells were incubated at 37°C and 5% CO₂ for 1 hour, fixated using 1% PFA. Mean fluorescent intensities (MFIs) of neutrophils (singlets>size>CD15⁺+CD16⁺) were assessed.
In a separate series of experiments, isolated healthy neutrophils were added to poly-L lysine coated Ibidi μ-slides and treated with plasma (20% final concentration) and/or inhibitors as described above. Neutrophil granules, released vesicles and NETS were stained using antibodies against myeloperoxidase (MPO, R&D Systems, AF3667) and neutrophil alkaline phosphatase (ALPL, MilliporeSigma, HPA008765) and secondary antibodies (anti-goat AF594, anti-rabbit AF647, 1:200, Invitrogen) along with 4′,6-diamidin-2-phenylindole (1:1000) and SytoxGreen (Thermo Fisher Scientific, S7020, 500 nM final concentration, see Supplemental Figure 4C).