Annexin 5

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.

Following treatment of Tet-inducible TUSC2 H1299 clone with 1 μg/ml doxycycline, 0.8 μM AF, and 0.8 μM erlotinib, cells were harvested, washed with PBS, and stained with Annexin-V (Promega, Madison, WI). Apoptotic activity was measured by flow cytometry using FlowJo software (Tree Star Corp, Ashland, OR). Values represent the mean of three independent experiments, each in quadruplicate.