Envision 2104 system

Ester lipids, plasmalogens and ferrostatin (fer-1) were added into 150 µL PBS as indicated to achieve 150, 150, and 9 ppm, respectively. Freshly dissolved 1.875 µM BODIPY 581/591 C11 in 150 µL PBS and 7.5 mM 2,2′-Azobis(2- amidinopropane) dihydrochloride (AAPH, VWR International) in 150 µL PBS were separately added to start the oxidation. PBS containing the same ratios of ethanol/methanol/DMSO served as control. After mixing thoroughly, reaction samples were incubated in the dark for 30 min at room temperature. 100 µL sample per well was measured using an Envision 2104 System (PerkinElmer) in black 96-well plates as triplicates. Fluorescence intensity at excitation 495 nm/emission 520 nm was evaluated and normalized to ethanol/methanol/DMSO control. Ferrostatin-1 was used as an antioxidant positive control.

SYK activation in iPSdMiG was determined as the ratio of phosphorylated SYK over total SYK. Therefore, 2 × 10⁴ iPSdMiG were seeded per well of a PLL-coated 96-well plate (Corning). After 24 h the cells were co-stimulated for 5 min with anti-TREM2 (5 µg/ml, R&D Systems, see Table 2) and respective CD33 antibodies or isotype controls (10 µg/ml, see Table 1). Thereby, a new P67.6 full-length antibody (BioLegend) was used as high endotoxin contamination was measured for the P67.6 Santa-Cruz antibody and possible effects of endotoxins on phosphorylation of SYK in iPSdMiG could not be excluded. pSYK levels were detected by the AlphaLISA SureFire Ultra p-SYK (Tyr525/526) Assay Kit (PerkinElmer) and normalized to the values of total SYK using the AlphaLISA SureFire Ultra Total SYK Assay Kit (PerkinElmer) according to the 2-plate assay protocol for adherent cells. All samples were measured as technical duplicates in 384-well OptiPlates (PerkinElmer). The plate was measured using the standard AlphaLISA settings on a PerkinElmer EnVision 2104 system. For the analysis, the technical duplicates were averaged and the pSYK signal was normalized to the total SYK signal. Data was displayed as pSYK/tSYK relative to the control anti-TREM2/IgG1 (anti-CD33 Ctrl).