Mab91671

Western blot analysis was performed on patient samples (plasma and TAs) for AAT and SLPI and NE from SARS-CoV-2 ARDS patients (plasma samples n = 20, TA samples n = 15) along with a control group samples from nsARDS patients (plasma samples n = 8, TA samples n = 6). Total protein quantities were measured with a BCA Protein Assay Kit (Pierce®). HRP-coupled primary antibodies for AAT (1:2000, GR286990-3, Abcam), NE (1:1000, clone # 950317, MAB91671, R&D systems) and SLPI (1:2000, AF1274, R&D systems, RRID: AB_2302508), were incubated overnight at 4 °C with the target protein, then probed with a secondary HRP-linked antibody (1:1000, 7076S, Cell signalling technology, RRID: AB_330924, and 1:1000, SC2020, Santa Cruz biotechnology, RRID: AB_631728, respectively). Proteins were detected with Immobilon ECL Ultra Western HRP Substrate (Merck Millipore) using a ChemiDoc™ Imaging System (Bio-Rad). Semi-quantification by densitometry was performed with ImageJ software (NIH).

Human neutrophils from healthy donors were isolated using Lympholyte®-poly solution (CL5070; Cedarlane) and stimulated with supernatants (500 μL) harvested from HT29 cells, either untreated (control; serum-free DMEM media) or treated with LPS ± LL-37 (for 4 h). Immunofluorescence analysis was performed as described above, using an anti-neutrophil elastase antibody (5 μg/mL; MAB91671; R&D Systems) and secondary Alexa549-conjugated donkey anti-mouse IgG antibody. For neutrophil elastase secretion quantification, fluorescence intensity was calculated using ImageJ 1.50i software. Average fluorescence intensity per replicate was quantified in five randomly selected fields of view and represented as fold increase in MFI.

Neutrophils (1 × 10⁶ cells) in the RPMI 1640 medium with 2% FBS were cultured for 4 h on poly-L-lysine-coated coverslips in the presence of 2.5 µM SYTOX green. Subsequently, the cells were fixed in 4% paraformaldehyde for 20 min; the supernatant was removed gently, and the cells were washed twice with PBS. Cells were permeabilized with 0.3% triton X for 10 min and blocked with 2% BSA for 1 h, followed by incubation with anti-MPO human/mouse polyclonal antibody (R&D System, #AF3667, 2:100), or anti-human neutrophil elastase (R&D System, #MAB91671, 1:100) overnight at 4 °C. The cells were washed with PBS and incubated with secondary antibodies coupled with fluorophores: donkey anti-goat Alexa Fluor 594 antibody (Thermo Fisher Scientific, #A-11058, 1:400) or chicken anti-mouse Alexa Fluor™ 594 (Thermo Fisher Scientific, #A-11058, 1:400) for 1 h at RT in the dark. Cells were then incubated with DAPI for 10 min and were visualized with an Olympus IX81 inverted microscope (Shinjuku City, Tokyo, Japan).

NET identification in tissue samples was performed by immunofluorescence staining. An anti-NE antibody (MAB91671) was purchased from R&D Systems (Minneapolis, USA) (1:50 dilution). An anti-H3Cit antibody (ab5103) was obtained from Abcam (Cambridge, UK) (1:50 dilution). The NE/H3Cit pair was researched in paraffin-embedded, 3-μm-thick sections. The slides were incubated with the primary antibodies at 4°C overnight after blocking with goat serum. Then, the sections were incubated with secondary antibodies (Alexa Fluor 488, green; and Alexa Fluor 647, red) (Abcam, Cambridge, UK) for 1 h at room temperature. DAPI was used for nuclear staining (ZSGB Biotech, Beijing, China). Finally, the slides were analyzed with a confocal laser scanning microscope (TCS-SP5; Leica, Wetzlar, Germany). For each specimen, the position of tumor and paratumor tissue was determined according to the results of H&E staining. Next, the average numbers of NE and H3Cit double-positive cells of tumor and paratumor tissues were calculated by two researchers counting five random 630x microscopic fields, respectively.