Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 1. Microglial TREM2 is required for the reduction in poly-GA proteins (A) GO analysis of the biological processes and molecular functions of upregulated differentially expressed genes in poly-GA-expressing cortices. Three bio- logical repeats were performed.

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Expressing

Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 3. Activation of the NLRP3 inflammasome inhibits poly-GA clearance by promoting TREM2 cleavage (A) Fluorescence images of poly-GA proteins in NLRP3 and TREM2 double- or single-knockout motor cortices. (B) Quantification of the area of GFP-GA signal in (A). n = 3, 3, 3, and 5 mice in each group; at least 3 cortex sections per mouse for statistics. (C) Immunoblot of mature TREM2 levels. BV2 cells were treated with IL-1b (10 ng/mL) for 12 h. (D) Immunoblot of cleaved TREM2 (sTREM2) levels in the cell medium of BV2 cells. (E) Immunoblotting showed that GI 254023X inhibited sTREM2 production. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/ mL, 12 h). (F) ELISA of sTREM2 levels in cerebrospinal fluid of control and poly-GA mice. n = 3 mice per group. (G) Immunoblot of p-DAP12 levels. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/mL, 12 h). Cell lysates were immu- noprecipitated by the anti-DAP12 antibody and immunoblotted with the antibody 4G10. (H and I) Quantification of immunofluorescent area of poly-GA. Wild-type or TREM2/ primary microglia were pretreated with GI 254023X (10 mM) and IL-1b (10 ng/mL) and incubated with FLAG-GA-His aggregates for 12 h totally. n = 3 independent experiments. (J) Immunoblot of active ADAM10. ATP1A1 indicated the loading control of BV2 cell membrane. Unless otherwise specified, at least three independent experiments were performed. t test in (F), (H), and (I). One-way ANOVA in (B).

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Activation Assay, Fluorescence, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Membrane

Journal: Biosensors

Article Title: Plasmonic Interferometers as TREM2 Sensors for Alzheimer’s Disease

doi: 10.3390/bios11070217

Figure Lengend Snippet: Design for TREM2 sensor chip based on plasmonic interferometry. ( a ) Cross-section schematic of the groove-slit-groove (GSG) architecture, which shows a slit flanked by two grooves, from which SPPs are excited by light diffraction and propagate towards the slit aperture, where they interfere and are then transmitted back into free space for far-field detection; diagram includes an example of an antigen complex, further described in . The bottom slab represents quartz, the middle titanium, and the top layer gold. ( b ) Scanning electron micrograph (SEM) of a GSG interferometer with p 1 = 7.65 μ m, p 2 = 8.15 μ m. ( c ) Schematic of plasmonic interferometer sensor chip layout. The chip contains four nominally identical sensing spots enabling multiplex sensing applications. The yellow area indicates quartz covered by gold and the blank area is an uncoated quartz window used for optical alignment. ( d ) Schematic of a representative active sensing area. Each sensing area contains two columns of single slits and two columns of nominally identical asymmetric GSG interferometers with separation distance of 300 μ m. The slit/grooves in each interferometer are ∼20 μ m long and, within each column, the distance between two adjacent interferometers is ∼40 μ m.

Article Snippet: Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in v. Finally, human TREM2 biotinylated antibody was purchased from R&D (Cat. #BAF1828, R&D) with 50 μ g bovine serum albumin (BSA) per 1 μ g as a carrier protein; the yielded solution contains 0.5% BSA in PBS.

Techniques: Multiplex Assay

Journal: Biosensors

Article Title: Plasmonic Interferometers as TREM2 Sensors for Alzheimer’s Disease

doi: 10.3390/bios11070217

Figure Lengend Snippet: Surface immobilization protocol for capture of TREM2 in solution. Chip surface was treated with ( i ) an RCA1 cleaning procedure followed by ( ii ) (3-Aminopropyl)triethoxysilane (APTES) to form an amino-terminated surface. Sulfo-NHS-biotin (sulfo-N-Hydroxysulfosuccinimide biotin) covalently attaches to the amino groups of the surface ( iii ) and subsequently captures streptavidins ( iv ). Finally, the streptavidin functionalized chip is bound by the biotinylated TREM2 antibody ( v ) for sensing of the TREM2 molecule ( vi ). The green dot in ( v ) represents the sulfo-NHS ester of biotin that acts as the biotinylation reagent and allows to form a stable bond between the antibody and the streptavidin already bound to the sensor surface, as reported in ( iv ).

Article Snippet: Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in v. Finally, human TREM2 biotinylated antibody was purchased from R&D (Cat. #BAF1828, R&D) with 50 μ g bovine serum albumin (BSA) per 1 μ g as a carrier protein; the yielded solution contains 0.5% BSA in PBS.

Techniques:

Journal: Biosensors

Article Title: Plasmonic Interferometers as TREM2 Sensors for Alzheimer’s Disease

doi: 10.3390/bios11070217

Figure Lengend Snippet: Tracking functionalization steps through plasmonic interference spectra. Measured results of transmitted intensity spectra after ( i ) RCA1, ( ii ) APTES, ( iii ) sulfo-NHS-biotin, ( iv ) streptavidin, and ( v ) biotinylated TREM2 antibody treatment. Solid lines represent the mean value of normalized intensity spectra averaged over seven nominally identical GSG interferometers after each functionalization step, as illustrated by the lower left insets. Light gray areas represent standard deviation. The vertical dashed line indicates the position of a representative transmission peak (588.1 nm) that results from constructive SPP interference after RCA1 cleaning. The black arrows mark the wavelength shift ( Δ λ ) in this reference peak as the result of new constructive interference conditions after each functionalization step.

Article Snippet: Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in v. Finally, human TREM2 biotinylated antibody was purchased from R&D (Cat. #BAF1828, R&D) with 50 μ g bovine serum albumin (BSA) per 1 μ g as a carrier protein; the yielded solution contains 0.5% BSA in PBS.

Techniques: Standard Deviation, Transmission Assay

Journal: Biosensors

Article Title: Plasmonic Interferometers as TREM2 Sensors for Alzheimer’s Disease

doi: 10.3390/bios11070217

Figure Lengend Snippet: Sensing temporal evolution of TREM2 surface binding kinetics with plasmonic interferometry. Blue circles represent the mean peak shift ( Δ λ ) averaged over seven nominally identical GSG plasmonic interferometers as the result of temporal evolution of antigen-antibody binding reaction for a 2.7 ng/ml TREM2 0.5% BSA PBS. Error bars represent the standard deviation. Bottom right inset illustrates the normalized transmitted spectra (averaged over seven identical GSG interferometers) measured at each time step. Color changing from dark red to yellow represents increasing reaction time from 0 to 60 min.

Article Snippet: Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in v. Finally, human TREM2 biotinylated antibody was purchased from R&D (Cat. #BAF1828, R&D) with 50 μ g bovine serum albumin (BSA) per 1 μ g as a carrier protein; the yielded solution contains 0.5% BSA in PBS.

Techniques: Binding Assay, Standard Deviation

Journal: Biosensors

Article Title: Plasmonic Interferometers as TREM2 Sensors for Alzheimer’s Disease

doi: 10.3390/bios11070217

Figure Lengend Snippet: Binding times for different TREM2 concentrations. Temporal evolution of peak wavelength shifts measured from normalized transmission spectra for different TREM2 concentrations. Error bars represent standard deviation from 7 GSG interferometers. Dashed lines represent exponential fit using the kinetic model provided in the text.

Article Snippet: Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in v. Finally, human TREM2 biotinylated antibody was purchased from R&D (Cat. #BAF1828, R&D) with 50 μ g bovine serum albumin (BSA) per 1 μ g as a carrier protein; the yielded solution contains 0.5% BSA in PBS.

Techniques: Binding Assay, Transmission Assay, Standard Deviation

Journal: Scientific reports

Article Title: Naturally-aged microglia exhibit phagocytic dysfunction accompanied by gene expression changes reflective of underlying neurologic disease.

doi: 10.1038/s41598-022-21920-y

Figure Lengend Snippet: Figure 4. Expression of TREM2, an Aβ42 receptor, is decreased in aging microglia. (a) Heatmap representation of phagocytic receptor gene expression levels for Young Aβ42+, Young Aβ42−, Old Aβ42+, and Old Aβ42−. The scale represents the row Z-score from 2 (highest expression) to − 2 (lowest expression). Young (2 months) and aged (21 months) microglia were incubated with 0.5 μM Aβ42 for 1 h and stained for microglial markers and TREM2 (n = 3/group; replicated 4 times with both male and female mice) (b) Representative flow plots (n = 3/ group) of young and aged microglia TREM2 expression and graph of % of young and old microglia that are TREM2+. (c) Representative flow plots of young and aged microglia TREM2 expression and Aβ42 uptake. (d) Graphs showing the average percentage of TREM2 expression in young (black) and old (red) Aβ42+ microglia and (e) Average percentage of Aβ fluorescence in young (black) and old (red) TREM2+ microglia. (f) Geometric mean fluorescence of Aβ42 in TREM2+ and TREM2− Aβ42+ microglia. *p ≤ 0.05, **p ≤ 0.01, mean ± s.e.m, Student’s t Test.

Article Snippet: After microglial enrichment and/or phagocytosis assay, samples were stained for flow cytometry using a fixable LiveDead viability stain (ThermoFisher Scientific) and surface stained with the following antibodies: anti-CD45 (clone: 104), -CD11b (clone: M1/70) (Biolegend), and -TREM2 (clone: 237920) (R&D Systems).

Techniques: Expressing, Gene Expression, Incubation, Staining, Fluorescence