af1828  (R&D Systems)

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: TREM2 agonism induces PLC activity in iPSC- derived macrophages. ( A ) Representative Western blot showing phosphorylation of SYK after TREM2 stimulation for 5 min using a TREM2-specific antibody (AF1828, R&D Systems) in Parent and TREM2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 6. ( B ) Ca 2+ flux induced by TREM2 ligation in Parent cells is absent in TREM2 Ko cells. n = 4. ( C) SYK inhibitor BIIB-057 (SYKi) reduces TREM2 antibody-evoked Ca 2+ signal in a dose-dependent manner in Parent cells. n = 3. ( D) TREM2-induced IP1 accumulation determined by HTRF assay is prevented in TREM2 Ko cells. n = 3, data shown represent mean ± SEM, ( B , D ) two-way ANOVA followed by Bonferroni’s multiple comparison test, ( C ) one-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Activity Assay, Derivative Assay, Western Blot, Phospho-proteomics, Ligation, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 Ko leads to dysregulation of cell surface marker expression and morphological changes in iPSC-derived macrophages. ( A ) Western blot confirms lack of PLCγ2 protein in PLCγ2 Ko and shows reduction in TREM2 expression in PLCγ2 KO macrophages compared to Parent. n = 4. Full immunoblot images are presented in Supplementary Fig. 7. ( B ) Levels of soluble TREM2 detected in cell supernatant 8 days after plating are lower in PLCγ2 Ko cells compared to Parent. n = 4, ( C ) Macrophage surface markers CD11b, CD14, and CD45 were measured by flow cytometry, compared to relevant isotype IgG. Annotations indicate frequency of marker positivity in the Parent line. ( D) Morphology of macrophage lines was determined by phalloidin staining and analysis of cell roundness and cell area. Scale bar 50 μm. n = 4, data shown represent mean ± SEM, One-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Marker, Expressing, Derivative Assay, Western Blot, Flow Cytometry, Staining, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 regulates TREM2-mediated signalling in iPSC- derived macrophages. ( A ) Representative Western blots showing phospho-SYK after TREM2 ligation using AF1828 in Parent and PLCγ2 Ko cells. ( B ) Quantification of SYK phosphorylation after TREM2 ligation shows no effect of PLCγ2 deficiency, n = 4. Full immunoblot images are presented in Supplementary Fig. 9. ( C ) pSYK HTRF assay confirms similar phosphorylation levels upstream of PLCγ2 upon TREM2 stimulation in Parent and PLCγ2 Ko cells, n = 3. ( D ) Ca 2+ flux induced by TREM2 ligation is prevented in PLCγ2 Ko cells. n = 3 ( E ) PLCγ2 deficiency abolishes TREM2-induced IP1 production as determined by HTRF assay. n = 3 ( F ) Representative Western blots and ( G ) quantification of ERK1/2 phosphorylation shows no major difference between Parent and PLCγ2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 10. Data shown represent mean ± SEM, two-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Derivative Assay, Western Blot, Ligation, Phospho-proteomics, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: Primary antibodies used for Western blot.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Western Blot