Journal: Brain, behavior, and immunity

Article Title: Profiling TREM2 expression in amyotrophic lateral sclerosis.

doi: 10.1016/j.bbi.2023.01.013

Figure Lengend Snippet: Fig. 2. TREM2 mRNA expression profiling in human spinal cord in ALS. (A) The figure illustrates the maps of the 3 different TREM2 transcript variants. Black boxes represent exons, and arrows represent transcription start sites. (B) Overall TREM2 mRNA levels in the spinal cord in ALS samples (n = 21) were compared with controls (n = 19) as shown in the graph. (C) The dot-plot graph shows mRNA expression of the 3 TREM2 variants in human spinal cord specimens. (D–F) The panels show mRNA expression levels in ALS patients when compared with controls for each of the TREM2 transcript variant. Data represent the mean value ± SD. *p value < 0.05; **p value < 0.01; ***p value < 0.001; ****p value < 0.0001.

Article Snippet: CSF and serum levels of sTREM2 in ALS patients and controls were determined by a commercial enzyme-linked immunoabsorbent assay (ELISA), the Human TREM2 DuoSet ELISA (Catalog number: DY182805; R&D systems, Minneapolis, MN, USA) according to manufacturer’s instructions.

Techniques: Expressing, Variant Assay

Journal: Brain, behavior, and immunity

Article Title: Profiling TREM2 expression in amyotrophic lateral sclerosis.

doi: 10.1016/j.bbi.2023.01.013

Figure Lengend Snippet: Fig. 3. TREM2 protein expression is increased in spinal cord from ALS patients by western blot analysis. (A) Spinal cord samples from controls and ALS patients were loaded as labeled on top of lanes. β-actin expression is shown as reference control. (B) The bar chart represents the quantitative measurement of TREM2 relative to β-actin protein expression (n = 10). Data represent the mean value ± Standard Error of the Mean (SEM). **p value < 0.01.

Article Snippet: CSF and serum levels of sTREM2 in ALS patients and controls were determined by a commercial enzyme-linked immunoabsorbent assay (ELISA), the Human TREM2 DuoSet ELISA (Catalog number: DY182805; R&D systems, Minneapolis, MN, USA) according to manufacturer’s instructions.

Techniques: Expressing, Western Blot, Labeling, Control

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: A – C Expression of M1 markers CD11c, CD86, and CD9 and D , E M2 markers CD206 and CD301 by ATMs of induced CLS and interstitial macrophages. F , G Expression of CD64 (all macrophages) or TREM2. Targeted adipocytes are marked by an asterisk. M2 ATMs outside of CLS are highlighted by arrows. Scale bars = 50 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: Whole mount antibody staining of CLS formed in vivo in lean mice under homeostatic conditions. A – C ATMs in in vivo formed CLS express pro-inflammatory markers CD11c, CD86, and CD9, while interstitial ATMs are negative (highlighted by arrows). D , E Interstitial ATMs express the anti-inflammatory markers CD206 and CD301 (highlighted by arrows), while ATMs in CLS are negative. F , G Expression of CD64 (all macrophages) or TREM2 (highlighted by arrows). Asterisks mark adipocytes inside CLS. Scale bars = 100 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Staining, In Vivo, Expressing

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