Sytox red

One hundred microliters of heparinized blood in the presence or absence of E. coli (3*10⁸/ml) were incubated for 1 h at 37°C. Then, an anti-human FITC-MPO antibody (20 µl/test) and SYTOX Red (10 nmol/L; Biolegend, USA) were added and incubated for 30 min at room temperature. Images were acquired on an ImageStream Multispectral Imaging Flow Cytometer (Amnis, part of EMD Millipore, USA) using the 40× magnification objective, which provides a numerical aperture (NA) of 0.75 and a pixel dimension of 0.5 m × 0.5 m.
A core diameter of 7 μm was used to maximize in-focus events. The 488 nm and 642 nm excitation laser were used at an output power of 150 mW. Objects with a minimum cross-sectional area of 50 m² and a maximum of 600 m² were collected to avoid acquiring debris or cellular aggregates. Typical files contained images of 20,000 cells. Cell images were analysed using IDEAS software, version 6.2. Cells in best focus were selected using the feature Brightfield (BF) Gradient RMS, a measurement of image contrast that excludes out-of-focus events. Doublets, aggregates, dead cells, and debris were excluded using SSC intensity and Syto intensity, and all analyses were restricted to single cells.

Hopx^CreER/Rosa^Td mice were treated with 2.5% DSS in drinking water for 7 days before switch to regular water. Hopx-tdTomato+ cells were labeled by a single injection of tamoxifen 13 days after the cessation of DSS treatment. Twenty-four hours after tamoxifen injection, the most distal 1cm section of colon was removed, opened, and treated with 30mM EDTA for 20minutes at 37°C followed by vigorous pipetting to enrich for crypts in the suspension. Single cell dissociation was then performed using an enzymatic cocktail containing 0.2mg/mL DNase1 (MilliporeSigma), 5mg/mL Dispase 1 (MilliporeSigma) and 2mg/mL Collagenase I (ThermoFisher Scientific) in the 37°C shaker at 250rpm. The cell suspension was incubated with EpCAM-BV421 (Biolegend) and Sytox-Red (Biolegend), which was used to gate for live epithelial cells during sorting (MoFlo). Sorted tdTomato+ epithelial cells were plated in Matrigel supplemented with 50% L-WRN medium and cultured for 6-7 days to count for spheroid formation efficiency.

Whole blood samples (100 μl) were stimulated with opsonized E. Coli (3*10⁸/ml), fMLP (0.83 µmol/L) or PMA (1.35 µmol/L). An anti-human FITC-MPO antibody (20 µl/test) and SYTOX Red (10 nmol/L) (Biolegend, USA) were added and the cells incubated for 30 min at room temperature. After lysis of the erythrocytes, the cells were analyzed using flow cytometry (Canto-II, BD, USA). Neutrophils were selected by FSC and SSC, and NET formation was defined as MPO and SYTOX Red double-positive, as previously described (19 (link)). Healthy human neutrophils were isolated (1*10⁶), and co-cultured separately with plasma from healthy volunteers (100 μl) or ACLF patients (100 μl) for 3 h for determining the effect of plasma on neutrophil NET formation. Hereafter, the samples were stimulated with opsonized E. Coli (3*10⁸/ml) for 1 h and were tested the neutrophil NET production by the above method.