Alphalisa surefire ultra total syk assay kit

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).