Mab1187

Spontaneous apoptosis of mature Trem1^+/+ versus Trem1^−/− neutrophils was assessed at 5 days post differentiation in SCF-containing medium and 8 h, 24 h, and 48 h later. Immediately upon removal from plates, neutrophils were washed in cold PBS and AnnexinV Binding Buffer and stained with FITC-conjugated AnnexinV (BD Pharmingen) according to the manufacturer's instructions. DAPI (Sigma-Aldrich) was added immediately prior to aquisition by flow cytometry. Only AnnexinV and DAPI double-positive cells (AnnexinV⁺ DAPI⁺) were considered in the analysis of apoptotic cells. For determination of TREM-1-mediated effects on apoptosis induction, 5 days differentiated neutrophils were stimulated in vitro in 96-well U-bottom plates at 2×10⁵ cells/well in complete RPMI medium lacking SCF in the presence of 10 µg/ml plate-bound anti-TREM-1 mAb (MAB1187; R&D) or a respective isotype control (RTK2758, Biolegend) for 24 h. Since the relative stickiness of neutrophils activated by plate-bound anti-TREM-1 did not allow for removal from the plates and FACS-based analysis of AnnexinV⁺ DAPI⁺ cells without causing a substantial bias by the selective analysis of non-adherent cells, Caspase 3/7 activity was determined by the ApoTox-Glo assay (Promega) according to manufacturer's instructions.

NK cell–mediated cancer cell growth was measured by a lactate dehydrogenase (LDH) detection assay kit (TaKaRa Bio Inc.) according to the manufacturer's protocol. The target cells, B16F10 mouse melanoma, were mixed with effector cells, splenic NK cells from WT mice or ApoE KO mice, in U-bottomed 96-well plate at the different NK cell/cancer cell ratios. In agonist and antagonist experiment, WT NK cells were pretreated with LP-17 peptide (antagonist, 100 ng/ml, Peptron) or anti-TREM-1 mAb (agonist, 10 μg/ml, MAB1187; R&D systems) for 24 h. Then co-cultured with B16F10 cells as target cells for 4 h, the supernatant was collected by centrifugation and transferred to flat-bottomed 96-well plate. Supernatant was analyzed by LDH release (yellow tetrazolium as substrate). Samples were measured using a spectrophotometric microplate reader at 490 nm. The percentage of specific lysis was calculated as follows: (experimental release – target spontaneous release – effector spontaneous release)/(target maximum release – target spontaneous release) × 100% (31 (link)).

TLR4-deficient BALB/c and Myd88-deficient C57BL/6 mice were provided by the Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea). dextran sodium sulfate (DSS) (MP Biomedicals, Solon, OH, USA) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Thermo Fisher Scientific, Waltham, MA, USA) were used to induce colitis and analysis was performed as previously described (11 (link)). At the time of DSS or TNBS treatment (day 0), we administrated an isotype control (IgG; R&D Systems, Minneapolis, MN, USA); three different α-TREM-1 (4 or 20 μg/mouse based on a previous study) (7 (link)); MAB1187 (R&D Systems) for experiments in C57BL/6 mice (Figure 1); AF1187 (R&D Systems) for all experiments, except those in Figure 2; or sc-19312 (Santa Cruz Biotechnology, Dallas, TX, USA) for the indicated experiments in BALB/c mice in Figure 2. All experiments using animals were approved by the Institutional Animal Care and Use Committee of Yonsei University Severance Hospital, Seoul, Korea (Approval No: 2014-0299).
The detailed methods for disease activity index (DAI) evaluation, histological analysis and immunohistochemistry, depletion or transfer experiments of microbiota, and metagenome analysis of microbiota are described in Supporting Information.