antibody baf1729 biotechne  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lipid associated macrophage marker genes are increased in obese tumor-adjacent mammary adipose tissue (mAT Tum-adj ) and positively correlate with tumor mass. B) Schematic for magnetically sorting CD11b + cells from E0771 tumors using Miltenyi Octomacs. C) Trem2 , Cd36 , and Cd9 relative gene expression by RT-PCR in tumor CD11b + fraction. C) Anatomical schematic of subcutaneous mAT Tum-adj and contralateral mammary adipose tissue (mAT Contra ). D - F) Trem2 , Plin2 , Cd36 relative gene expression by RT-PCR from whole tissue lysates of mAT Contra and mAT Tum-adj . G-H) Correlation analysis of tumor mass vs. Trem2 and Plin2 gene expression in mAT Contra and mAT Tum-adj with linear regression fit. Figures D-F, solid bar = mAT Contra , hashed bar = mAT Tum-adj . One-way ANOVA tests were used to compare tumor LAM marker gene expression among groups. Two-way ANOVA with Tukey’s multiple comparison tests were used to compare groups for all mAT gene expression. Simple linear regression analysis was applied to correlation plots and the p value and coefficient of determination (r 2 ) is displayed on each graph. All data plotted as mean ± SEM for 8-12 mice per group. *denotes diet effect, †denotes tissue effect (mAT Contra vs. mAT Tum-adj ), **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, †††p<0.001, ††††p<0.0001. & C were created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Trem2 ablation attenuates tumor growth, decreases lipid associated macrophages, and increases adipocyte hypertrophy in a chow-fed postmenopausal breast cancer model. A) Schematic of study design for chow-fed postmenopausal breast cancer model in Trem2 +/+ and Trem2 -/- mice. B) sTREM2 (ng/mL) in plasma from female Trem2 +/+ and Trem2 -/- mice measured by ELISA. C) Blood glucose (mg/dL) measured during 14-week time point intra-peritoneal glucose tolerance test (ipGTT) and corresponding integrated area under the curve (iAUC). D) Tumor volume (mm 3 ) over time measured every 1-2 days. E) Tumor mass (g) at study endpoint. F) Relative gene expression by RT-PCR of LAM and macrophage markers from oAT whole tissue lysates. G) Adipocyte sizing frequency in tumor-adjacent mammary adipose tissue (mAT Tum-adj ). Diameter: small = 25-49μm, medium = 50-69μm, large = 70-99μm, very large = 100-150μm, extremely large = >150μm. H-I) Representative image of Trem2 +/+ and Trem2 -/- mAT Tum-adj stained with hematoxylin and eosin. Scale bar represents 100μm. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare blood glucose over time in the ipGTT. Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes. Unpaired two-tailed t-tests were used to compare groups for sTREM2 ELISA, iAUC, tumor mass, LAM gene expression, and each binning diameter size in adipocyte sizing analysis. All data plotted as mean ± SEM for 10-13 mice per group. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. was created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lean, but not obese or weight loss, Trem2 -/- mice exhibit decreased tumor growth. A) Body weight (g) measured weekly with dashed lines to indicate time of diet switch and E0771 PD-L1 cell injection. For diet groups and genotypes, light green = lean Trem2 +/+ , dark green = lean Trem2 -/- , light blue = obese Trem2 +/+ , dark blue = obese Trem2 -/- , light pink = weight loss Trem2 +/+ , and maroon = weight loss Trem2 -/- . B) sTREM2 (ng/mL) in plasma from Trem2 +/+ and Trem2 -/- mice measured by ELISA.C) Tumor mass (g) at study endpoint. D-F) Tumor volume (mm 3 ) over time for lean, obese, and weight loss Trem2 +/+ and Trem2 -/- mice, respectively. G) Diagram of tumor and tumor-adjacent mammary adipose tissue CD45 + cell sorting and processing for single cell RNA Sequencing. Mixed effects analysis with uncorrected Fisher’s LSD test was used compare tumor volumes over time between genotypes in the lean group (one missing value in lean Trem2 +/+ group at day 28). Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes in obese and weight loss groups. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare tumor mass between genotypes in lean, obese, and weight loss groups. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ), †denotes diet effect, *p<0.05, **p<0.01, ****p<0.0001 for genotype differences (Trem2 +/+ vs. Trem2 -/- ) and ††††p<0.0001 for diet differences. All data plotted as mean ± SEM for n=4-23 per group. Figure G was created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, FACS, RNA Sequencing Assay, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Myeloid populations in tumors and tumor-adjacent mammary adipose tissues (mAT Tum-adj ) analyzed by single-cell RNA sequencing. A-B) Uniform Manifold Approximation and Projection (UMAP) of tumor (A) and mAT Tum-adj (B) myeloid cell subclusters from merged conditions labeled broadly by cell type category and colored by high-resolution cell type identities. C-D) Myeloid cell subcluster proportions expressed as frequency (%) split by diet-genotype groups for tumor (C) and mAT Tum-adj (D). E-F) Trem2 expression in lean, obese, and weight loss Trem2 +/+ mice in tumor (E) and mAT Tum-adj (F). G) Dot plot of the top 5 differentially expressed genes (DEGs) between lean Trem2 +/+ and lean Trem2 -/- tumor Macro_C1qc subclusters. Genes include H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 . Log fold change is shown for all 6 diet-genotype groups. H) Dot plot of H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 log fold change in the mAT Tum-adj LAM subcluster shown in all 6 diet-genotype groups.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: RNA Sequencing Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Visualization and binning of Tumor T cell clones reveals expansion of highly clonal CD4 + Th1 cells in lean Trem2 -/- tumors. A) Stacked bar graph of tumor T cell subclusters indicating cells per gram of tumor across all 6 diet-genotype groups. B) Tumor T cell subcluster proportions expressed as frequency (%) across all 6 diet-genotype groups. C) Uniform Manifold Approximation and Projection (UMAP) of tumor T cell subclusters from merged conditions colored by high-resolution cell type identities. D-I) Tumor T cell clones with a clonal population of two or more visualized by mapping clonal cell counts, indicated by color, onto the tumor T cell UMAP. The data is split as labeled by diet and genotype. J) Stacked bar graph showing the distribution of tumor T cell subclusters, binned as single cells or clonal low, clonal moderate, or clonal high (low = x ≤ 12 (median); moderate = 13 ≤ x ≤ 79 (3 rd quartile cutoff); high = x >79) shown for all 6 diet-genotype groups.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Clone Assay, Labeling

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Obese mice do not respond to αPD-1 therapy and do not exhibit an increase of T cell activation markers in tumor-adjacent mammary adipose tissue (mAT Tum-adj ) or tumor. A) Schematic of study design for lean, obese, and weight loss breast cancer model treated with IgG and αPD-1. During the last two weeks of tumor monitoring, mice were injected with either αPD-1 or IgG isotype control. B) Tumor mass (g) at study endpoint. C-E) Tumor volume (mm 3 ) over time for lean, obese, and weight loss IgG and αPD-1 treated mice with dashed line to indicate start of antibody treatment. F) Schematic of isolating the stromal vascular fraction from the mAT Tum-adj and subsequently isolating a pooled sample of CD4 + and CD8 + T cells using the magnetic Miltenyi Octomacs microbead sorting system. G) T cell activation markers, Pdcd1 , Tigit , Tox , and Lag3 relative gene expression by RT-PCR from magnetically sorted CD4 + and CD8 + T cells from the mAT Tum-adj . H) Cd8a , Pdcd1 , Lag3 , and Trem2 gene expression by RT-PCR from whole tumor lysates. Due to the response to αPD-1 treatment in the lean mice, only two tumors from the lean αPD-1 group could be analyzed by RT-PCR. For diet groups, solid colors: green = lean IgG, blue = obese IgG, pink = weight loss IgG; open circles: dark green = lean αPD-1, dark blue = obese αPD-1, maroon = weight loss αPD-1. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare groups for tumor mass. Mixed effects analysis was used for statistical analysis of tumor volumes over time. Two-way ANOVA with Sidak’s multiple comparison test was used to compare groups in the T cell activation marker gene expression analysis. All data plotted as mean ± SEM for 2-6 mice per group. *denotes antibody effect (IgG vs. αPD-1), †denotes diet effect, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, ††p<0.01. , F, & H were created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Activation Assay, Injection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Marker

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for alveolar macrophages and not shared with interstitial macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (b) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for interstitial macrophages and not shared with alveolar macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (c) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for classical monocytes and not shared with interstitial macrophages and alveolar macrophages. Data reanalyzed from previously published scRNA-seq data . (d) Venn diagram showing the overlap between hyperoxia-induced genes (FC > 2; FDR < 0.05) in 3 different myeloid cell subsets; alveolar macrophages (n = 281), interstitial macrophages (n = 275), classical monocytes (n = 215) (left panel). Venn diagram showing the overlap between hyperoxia-induced genes in the three myeloid cell subsets (n = 18) and whole lung tissues (n = 253 in ) (right panel). (e) SNPs in the Trem2 gene between B6 and DBA mice. (f) Body weight in hyperoxia-exposed WT (B6) (same samples as ) and T2KO mice. Data are mean ± SEM (n = 6 for each group).

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques:

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Single-cell RNA-seq analysis of whole lung tissues from B6 mice exposed to 95% oxygen from P0 to P5 . (b) Trem2 mRNA expression in the lungs of B6 and DBA mice. Bar graphs show mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p -adj < 0.01 and *** p -adj < 0.001. (c) Immunoblots for TREM2 protein in whole lung tissues collected on P14 from B6 and DBA mice. Bar graphs show mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01. Uncropped images of the blots are shown in . (d) Soluble TREM2 levels in the plasma of B6 and DBA mice on P14 after neonatal hyperoxia exposure. Bar graphs show mean ± SEM (n = 3-8 per group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. (e) H&E staining was performed to assess the alveolar complexity at P14 in WT (B6) mice (left panels) and T2KO mice (right panels) with scale bars denoting 100 μm. The bar graph represents the results of the quantification of alveolar simplification using mean linear intercept (same WT (B6) samples as ). Data are mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. (f) Bronchoalveolar lavage (BAL) was performed at P14 in WT (B6) and T2KO hyperoxia-exposed and normoxic control mice (n = 14 for WT (B6) and n = 11-13 for T2KO mice). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. n.s.: not significant. (g) Matrix ultrastructural analysis of lung tissues of neonatal hyperoxia-exposed B6 (WT), DBA and T2KO mice on P14. Manifold of hyperoxia-induced pathological architecture with higher pseudotime representing increasingly disrupted architecture and alveolar simplification. Tissue images show representative tiles along the pseudotime trajectory. (h) Architecture-defining parameters. Identification of top 5 individual matrix features associated with low pseudotime (normal-like interstitium) and high pseudotime (more aberrant interstitium), based on Pearson correlations. Parameters are displayed as the absolute value of the Pearson coefficient (i.e. magnitude of correlation, as shown by height of arrows on y-axis), with all p < 0.001. (i) Visualization of hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. Hyperoxia-exposed lungs from DBA and T2KO mice with less lung matrix remodeling localize near the root point of the pseudotime trajectory. (j) Bar graphs of matrix pseudotime for hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. The difference in pseudotime normalized to normoxic control lungs is shown. Data are mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. n.s.: not significant. Each dot represents one mouse (b, d, e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: RNA Sequencing Assay, Expressing, Comparison, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) BMDMs were obtained from B6 (WT), DBA and T2KO mice and exposed to hyperoxia (95%) for 24 h. (b) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed BMDMs obtained from B6 (WT), DBA and T2KO mice and the normoxic controls. Uncropped images of the blots are shown in . (c) RNA-seq of hyperoxia-exposed BMDMs from B6 (WT), DBA and T2KO mice and the normoxic controls. (d) Venn diagram showing the overlap between hyperoxia-induced genes in B6 (WT) BMDMs (n = 173) and hyperoxia-suppressed genes in T2KO (n = 570) and DBA (n = 905) BMDMs. Gene Ontology analysis of 34 genes induced by hyperoxia in B6 (WT) BMDMs and suppressed in T2KO and DBA BMDMs. (e) Bar plots for expression of representative genes belonging to the p53 signaling pathway. Data are mean ± SEM. DSeq2 was used for comparisons. ** p -adj < 0.01 and *** p -adj < 0.001. (f) Caspase 3 activity measured in cytosolic fractions from BMDMs from B6 (WT), DBA and T2KO mice. Data are mean ± SEM. (n = 4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. Each dot represents one mouse (e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot, RNA Sequencing Assay, Expressing, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed (1, 2, 4 or 24 h) BMDMs obtained from B6 (WT) mice and normoxic controls (0 h). Uncropped images of the blots are shown in .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 µl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 µg/ml for 90 minutes at room temperature.

Techniques: Clone Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 µl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 µg/ml for 90 minutes at room temperature.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 µl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 µg/ml for 90 minutes at room temperature.

Techniques: Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAP visualization of mouse hippocampal cell clusters classified by cell type based on DEG identified by Seurat v4. Violin plots represent the log-normalized expression of Itm2b and Trem2 across cell populations in mouse hippocampal cell clusters. ( B ) Human DFC cell clusters, classified by cell type as in ( A ). Violin plots represent the log-normalized expression of ITM2B and TREM2 across cell populations in human DFC cell clusters. ( C ) List of cell-type- specific marker genes used to annotate major brain populations. ( D ) Itm2b and Trem2 mRNA expression in mouse microglia and non-microglia cells analyzed by quantitative RT-PCR. Data information: The data sets analyzed are publicly available and are described in the two following papers: mouse scRNAseq data set (Zeisel et al, ), hippocampus n = 5 females, n = 5 males; human snRNAseq data set (Li et al, ), n = 10 (sex not specified). More information about these datasets can be found in the cited paper. Statistical comparisons between the groups shown in ( D ) was conducted using two-tailed unpaired t test **** P < 0.0001. The data are derived from are from 15-month-old w/w control animal, male n = 3, females n = 3; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Marker, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Control

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of objects Data 1 and Data 2 before filtering. ( B ) UMAPs of objects Data 1 and Data 2 after filtering. ( C ) UMAP of Object 1 after integration, filtering, and re-clustering, shows 16 microglia clusters. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. These cells derive from: Trem2-KO , 1 male and 1 female; Itm2b-KO , 2 males and 2 females; WT controls, 1 male and 2 females; Itm2b/Trem2-dKO , 1 male and 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques:

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of microglia grouped by genotype. ( B ) Average scaled expression levels of selected signature genes per cluster and cluster’s annotation based on expression of signature genes. ( C ) Volcano plots showing differentially expressed genes in clusters I/T-D1, 2, 3 and 4. ( D ) Proportional contribution of each genotype and proportional contribution of individual samples of each genotype to cluster 3. ( E ) KEGG pathway enrichment analysis of pathways upregulated in cluster 3. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. Volcano plots in ( C ) were obtained using Fast Wilcoxon rank sum test and auROC. These cells derive from: Trem2-KO , n = 1 male and n = 1 female; Itm2b-KO , n = 2 males and n = 2 females; WT controls, n = 1 male and n = 2 females; Itm2b/Trem2-dKO , n = 1 male and n = 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published. All data are expressed as means +/− SEM.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) N2A or HEK293 cells were transfected with F-BRI2 (B) and Trem2 (T), either alone (V=empty pcDNA3.1vector) or in combination and analyzed by Western blot with anti-FLAG (M2) and anti-Trem2 (NT1) on total lysates (T.L.) and M2 immunoprecipitants (IP-M2). Immunoprecipitants bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. For each cell line, two independent transfections were performed (Exp. 1 and Exp. 2). ( B ) Schematic representation of Trem2 and the two products of α-secretase cleavage, sTrem2, and Trem2-CTF. TM indicates the transmembrane region of Trem2. Red bars point to the antigenic regions used to produce the anti-Trem2 antibodies CT, NT1 and NT2. The cytosolic and intralumenal/extracellular regions of Trem2 are indicated. ( C ) Western blot analysis with anti-FLAG, anti-Trem2 NT1, and anti-Trem2 CT antibodies of T.L. and IP-M2 from HEK293 cells transfected with F-BRI2 and Trem2, either alone or in combination, with or without deglycosylation. *Indicates Trem2 species of unclear primary structure. Trem2 (f.l.) indicates full length Trem2. ( D ) Western blot analysis with anti-FLAG and anti-Trem2 CT antibodies of immunoprecipitants obtained with CT, NT1, and NT2 antibodies from HEK293 cells expressing either F-BRI2 alone or F-BRI2 plus Trem2. The nature of the bands migrating above 100 kDa in the NT2 IP samples is unknow. ( E ) Schematic representation of the F-BRI2 constructs used in ( F ). The Bri23 region, transmembrane region (TM), Brichos domain, APP-binding domain (APP BD), FLAG tag (F), cytosolic and intralumenal/extracellular regions are indicated. ( F ) WB analysis with anti-FLAG and anti-Trem2 antibodies of lysates and immunoprecipitants from HEK293 cells expressing F-BRI2 deletion mutants plus Trem2 or Trem2 alone (V). The * indicates Trem2 species of unclear primary structure. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Western Blot, Expressing, Construct, Binding Assay, FLAG-tag

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of the bicistronic expression plasmids used for HEK293 cell transfections in Panel ( B ): TREM2-3xFLAG + Myc-BRI2, 3xFLAG + Myc-BRI2 1-131 , 3xFLAG + Myc-BRI2 1-80 , and 3xFLAG + Myc-BRI2δ 80-131 . The TREM2-3xFLAG is expressed by the 5’ cistron, while BRI2 proteins are expressed by the 3’ cistron. ( B ) Western blot analysis using anti-FLAG, anti-Myc, and anti-human BRI2 antibodies of Total Lysate and Immunoprecipitation (M2-IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. The anti-human BRI2 antibody exhibits reactivity toward Myc-BRI2 and Myc-BRI2 1-131 suggesting recognition of an epitope located within the amino acids 80-131 region of BRI2. ( C ) Schematic representation of the bicistronic expression plasmids employed for HEK293 cell transfections in Panel ( D ): 3xFLAG-BRI2 + TREM2-Myc, 3xFLAG-BRI2 + TREM2-δIg-like-Myc, 3xFLAG-BRI2 + TREM2-CTF-Myc, 3xFLAG-BRI2 + TREM2-W198Ter-Myc, and 3xFLAG-BRI2 + TREM2-δ/α-site-Myc. The 3xFLAG-BRI2 is expressed by the 5’ cistron, while TREM2 proteins are expressed by the 3’ cistron. ( D ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of Total Lysate (T.L.) and Immunoprecipitation (IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. * Indicates protein signals of unclear nature. ** and *** indicate TREM2W198Ter signals of unclear primary structure. A longer exposure (Long Exposure) of the Anti-hTREM2-CT Western blot for the 3xFLAG-BRI2 + TREM2-Myc transfection revealed traces of TREM2-CTF precipitating with BRI2. Data information: This figure represents one of three independent experiments conducted. Data from the other two experiments are presented in Fig. . We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with anti-FLAG, anti-Myc antibodies, and anti-human BRI2 antibodies of total lysates and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( B ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of total lysates (T.L.) and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( C ) Co-immunoprecipitation of endogenous Bri2 and Trem2 from mouse primary macrophages. Samples were deglycosylated before Western blot. Data Information: Panels ( A ) and ( B ) represent two independent experiments conducted similarly to those in Figs. B and , respectively. Panel ( C ) shows the only co-immunoprecipitation of endogenous Bri2 and Trem2 performed to date. The complete membrane images used for Western blot analyses are included without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Immunoprecipitation, Transfection, Membrane

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of TREM2-ECD, sTREM2, BRI2-ECD and BRI2-BRICHOS recombinant proteins. TREM2-ECD encompasses the entire extracellular domain of TREM2, and BRI2-ECD encompasses the entire extracellular domain of BRI2, including the second putative TREM2-interacting domain. BRI2-BRICHOS and BRI2-ECD were fused with a 3xFLAG tag at their N-terminus, enabling immunoprecipitation using anti-FLAG M2-Agarose beads for the purification of protein complexes in a cell-free system via elution with a 3xFLAG peptide. The diagram highlights the signal peptides (SP), 7-Histidine tag (7-His, employed for protein purification), the 3XFLAG tag (3xF, utilized for complex purification), Ig-like domain (of TREM2), and BRICHOS domain (of BRI2). ( B ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated overnight at 4 degrees Celsius with M2-Agarose beads at a concentration of 2 μM for each protein. Following extensive washing, complexes bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. Unbound proteins and eluates (3xFLAG elu.) were analyzed by Western blot using either the anti-FLAG antibody M2 or an anti-human TREM2 N-terminal antibody (TREM2-NT). sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( C ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, 3xFLAG + sTREM2, 3xFLAG + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated as in B. Proteins eluted with the 3xFLAG peptide were analyzed by Western blot using either M2 or TREM2-NT. sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( D ) Western blot analysis using M2 and TREM2-NT antibodies of a new experiment mirroring the setup in ( B ). Eluates were separated under reducing and non-reducing conditions. BRI2-BRICHOS and BRI2-ECD monomers, dimers, trimers, and tetramers are indicated by the numbers 1, 2, 3, and 4, respectively. Higher multimolecular complexes are present but not labeled. The sTREM2 and TREM2-ECD bound to BRI2-BRICHOS and BRI2-ECD analyzed under non-reducing conditions show no significant increase in molecular weight compared to those analyzed under reducing conditions. ( E ) Decreasing concentrations (4, 2, 1, 0.5, 0.25, and 0 μM) of BRI2-BRICHOS and BRI2-ECD were incubated with 2 μM of either sTREM2 or TREM2-ECD and analyzed as described in ( C ). The bottom panel displays a Western blot of deglycosylated eluates using the TREM2-NT antibody. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Immunoprecipitation, Purification, Protein Purification, Incubation, Concentration Assay, Western Blot, Labeling, Molecular Weight

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) HEK293 cells were transfected with Trem2 and either empty vector (V) or F-BRI2 (B). NT are non-transfected cells. Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1. *Indicates Trem2 species of unclear primary structure. ( B ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( A ). ( C ) HEK293 cells were transfected with Trem2 and either empty vector (V), F-BRI2 or deletion mutant F-BRI2 1-80 . Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1 (lower panel). The * indicates Trem2 species of unclear primary structure. ( D ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( C ). ( E ) HEK293 cells were transfected with either Trem2 (T) or empty vector (V). Following transfection, lysates and media underwent deglycosylation and were subsequently analyzed by Western blot using anti-Trem2 antibodies CT and NT1. In the CT Western blot, the asterisk (*) indicates a Trem2-derived polypeptide that retains the CT epitope and is likely to lack part of the N-terminal Trem2 sequence, causing a reduction in size. In the NT1 Western blot, the double asterisk (**) highlights a Trem2-derived polypeptide that retains the NT1 epitope and is likely to lack part of the C-terminal sequence. The presence of this band primarily in cell lysates suggests potential retention of the transmembrane region and/or localization within intracellular compartments. Its low-level detection in the media further suggests intracellular origin. The band marked as sTrem2 is marked as sTrem2 because: (1) is of the expected size for deglycosilated sTrem2; (2) it is notably enriched in the media, consistent with the preferential localization of sTrem2 in extracellular fluids. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using two-tailed unpaired t test ( B ) and one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( C , D ). * P < 0.05, ** P < 0.01, *** P < 0.001. The data presented are derived from are from: Trem2+Vector transfectant n = 5, Trem2+F-BRI2 transfectant n = 5 ( A , B ); Trem2+Vector transfectant n = 3, Trem2+F-BRI2 transfectant n = 3, Trem2+F-BRI2 1-80 n = 3 ( C , D ); the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Derivative Assay, Sequencing, Two Tailed Test

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of ELISA 1 and ELISA 2. Both ELISAs use the same Biotinylated-αTrem2 capture antibody (in black). ELISA 1 uses αTrem2-CT (red) + Sulfo-αRabbit (blue) detection antibodies. ELISA 2 uses αTrem2-NT (orange) + Sulfo-αRat (green) detection antibodies. Trem2 can be detected by both ELISAs, sTrem2 can be detected only by ELISA 2: neither ELISA can detect Trem2-CTF. ( B ) Quantification of Trem2 and sTrem2 in the P100 and S100 brain fractions of ~245 days old w/w control, Itm2b-KO and Trem2-KO mice. ( C ) Western blot analysis of P100 fractions from a representative w/w, Trem2-KO and Itm2b-KO P100 sample with αTrem2-CT and an αBri2 antibody. *Indicates a non-specific band. ( D ) Detection and quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software; GAPDH was used as a loading control. ( E ) ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of w/w, Trem2-KO and Itm2b-KO animals. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. The membrane in ( C ) was cut at the 20 and 15 kDa molecular weight marker (MWM). The upper section was probed with the anti-Bri2 antibody, while the lower section was probed with the Trem2-CT antibody. Similarly, in ( D ), the two membranes were divided at the 20 and 15 kDa MWM. The upper portion was probed with the anti-Gapdh antibody, while the lower portion was probed with the Trem2-CT antibody. Statistical comparisons among the groups were conducted using one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( B , E ); two–way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( D ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from are from w/w control, females n = 7, males n = 12; Itm2b-KO females n = 6, males n = 7; Trem2-KO , females n = 6, males n = 7; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Software, Membrane, Molecular Weight, Marker, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) CD11b and CD45 staining, and FACS analysis of brain cells isolated from Cx3cr1 CreER/wt and Cx3cr1 wt/wt animals. ( B ) FACS analysis of sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from Itm2b f/f :Cx3cr1 CreER/wt animals. ( C ) Schematic representation of the PCR test used to identify the Itm2b f and Itm2b KO alleles. ( D ) PCR analysis of genomic DNA isolated from EYFP + and EYFP - cells sorted from Itm2b f/f :Cx3cr1 CreER/wt brains. ( E ) Analysis of Itm2b and Trem2 mRNA expression in sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from ~14 months-old Itm2b f/f :Cx3cr1 CreER/wt and Itm2b w/w :Cx3cr1 CreER/wt animals. ( F ) ELISA 2 was used to measure sTrem2 levels in Itm2b f/f :Cx3cr1 CreER/wt and Itm2b f/f :Cx3cr1 wt/wt littermates. Data information: Statistical comparisons among the groups were conducted two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( E ); two-tailed unpaired t test ( F ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from: (E) Itm2b f/f :Cx3cr1 CreER/wt , females n = 5, males n = 7; Itm2b w/w :Cx3cr1 CreER/wt , females n = 3, males n = 4; ( F ) Itm2b f/f :Cx3cr1 CreER/wt , females n = 15, males n = 12; Itm2b f/f :Cx3cr1 wt/wt , females n = 10, males n = 11; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the S100 brain fractions of ~245 days old control and FDD-KI mice. Data were analyzed by unpaired T -test. All data are shown as means +/− SEM: **** P < 0.0001. ( B ) Quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software. ( C ) Red Ponceau staining (upper panel) was used to normalize the Trem2-CTF signal (lover panel) obtained by Western blot. Data information: Statistical comparisons among the groups were conducted using unpaired T -test. **** P < 0.0001. The data presented are derived from: ( A ) w/w mice (females, n = 7; males, n = 12) and FDD-KI mice (females, n = 8; males, n = 9) mice; ( B ) w/w mice (females, n = 3; males, n = 3) and FDD-KI mice (females, n = 6; males, n = 7) mice; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Control, Western Blot, Software, Staining, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia using ELISA (left panel) and Western blot of deglycosylated conditioned media with Trem2 NT antibody (quantification of Western blot is shown in the second panel). Trem2 CT antibody does not show any signal. ( B ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 5 h of serum starvation by ELISA. ( C ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 1 and 2 h with either vehicle (Veh) or E. coli by ELISA. ( D ) Western blot of deglycosylated cell lysates from WT and Itm2b-KO primary microglia, 2 h after E. coli stimulation, with Trem2 CT antibody to visualize Trem2 f.l. and Trem2-CTF, along with quantification of the Trem2-CTF/Trem2 f.l. ratio. ( E ) Quantification of the Trem2-CTF/Trem2 f.l. ratio for only the two untreated (Veh) groups. ( F ) Analysis of Itm2b and Trem2 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( G ) A second set of biological replicates was analyzed following the same procedure as in panel ( C ). ( H ) A second set of biological replicates was analyzed following the same procedure as in panel ( D ). ( I ) A second set of biological replicates was analyzed following the same procedure as in panel ( F ). Data information: Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B , E , F , I ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C , D , G , H ). * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3 for ( A , C , D , E – I ), n = 6 for ( B )) and Itm2b-KO primary microglia ( n = 3 for Exp. and n = 3 for ( A , C , D , E – I ), n = 6 for ( B )); the letter “n” indicates biological replicates, except for ( B ), which includes 3 biological replicates with 2 technical replicates each. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with Trem2 CT antibody of deglycosylated cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the Trem2-CTF/Trem2 f.l. ratios from the Western blot shown in the right panel. ( B ) sTrem2 ELISA on conditioned media from these cell cultures (left panel). The right panel shows an ELISA performed using media from cells treated with vehicle and incubated before and during the ELISA with either vehicle (PBS) or 2 μM of BRI2-ECD. The evidence that incubation with BRI2-ECD does not change the ELISA quantification indicates that BRI2-ECD does not interfere with the quantification of sTrem2 by ELISA. ( C ) Western blot analysis with anti-Syk and anti-pSyk antibodies of cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the pSyk/Syk ratios from the Western blot is shown in the right panel. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using a two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001. The data presented are derived from Itm2b-KO primary microglia ( n = 3 for each condition); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Acta Neuropathologica Communications

Article Title: Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion

doi: 10.1186/s40478-024-01734-x

Figure Lengend Snippet: Aβ immunostaining and ELISA-based measurements of Aβ aggregates in brain tissue from tg-UppSwe, tg-ArcSwe and tg-Swe mice. A Immunostaining of tg-UppSwe, tgArcSwe and tg-Swe brain tissue sections (hippocampus, 40× magnification) with 3D6 (yellow, upper panel) or mAb158 (yellow, lower panel) in comparison with double staining using Aβ40 and Aβ42-specific antibodies (green). Scale bar: 200 µm. B ELISA quantification of total Aβ1-40 and 1-42 levels in FA brain extract. C 3D6-3D6 ELISA quantification of total Aβ aggregates in TBS 100K , TBS 16K and TBS-T brain extracts. D mAb158 positive fraction of the total Aβ aggregates detected by 3D6-3D6 ELISA in TBS 100K , TBS 16K and TBS-T brain extracts. E Distribution of [ 125 I]RmAb3D6-scFv8D3 and [ 125 I]RmAb158-scFv8D3, three days after administration of these bispecific antibodies at 32 nmol/kg body weight (therapeutic dose) to 18-month-old tg-UppSwe mice, expressed as a brain-to-blood radioactivity ratio in whole brain tissue (Brain) and in TBS 16K , TBS-T and FA extracts. F Soluble Aβ aggregates in TBS 100K and TBS 16 K brain extracts from 18 months old tg-UppSwe mice three days after administration of a therapeutic dose (32 nmol/kg) of [ 125 I]RmAb3D6-scFv8D3 or [ 125 I]RmAb158-scFv8D3, in comparison with PBS. Non-significant ( ns ), * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The TBS 16K and TBS-T brain extracts were diluted in ELISA incubation buffer (PBS, 0.1% BSA, 0.05% Tween) and incubated over night at 4 °C, followed by detection with 0.25 µg/ml biotinylated anti-TREM2 BAF1729 (R&D), HRP-conjugated streptavidin and K Blue Aqueous TMB substrate and read at 450 nm with a spectrophotometer.

Techniques: Immunostaining, Enzyme-linked Immunosorbent Assay, Comparison, Double Staining, Radioactivity

Journal: Acta Neuropathologica Communications

Article Title: Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion

doi: 10.1186/s40478-024-01734-x

Figure Lengend Snippet: Astroglial response to Aβ. A Aβ40 + 42 and GFAP immunostaining of brain tissue from 18-month-old tg-UppSwe, tg-ArcSwe and tg-Swe mice. Co-localization of Aβ and GFAP was lacking in tg-UppSwe mice but was abundant in tg-ArcSwe and tg-Swe mice. Scale bar 100 µm. ELISA quantification of GFAP in TBS ( B ) and TBS-T ( C ) brain extracts from 18-months-old wt, tg-UppSwe, tg-ArcSwe and tg-Swe mice. Not significant ( ns ), ** P < 0.01, *** P < 0.001. D Schematic description of the procedure to establish primary astrocyte monocultures originating from the cerebral cortices of embryonic mouse brain. Primary astrocyte cultures were exposed to sonicated, Cy3-labeled fibrils of synthetic Aβ Upp , Aβ Arc or Aβ wt (all Aβ1-42). Cells were stained for GFAP, LAMP-1 and cell nuclei (Dapi). While Aβ Upp clustered on the surface of cells, Aβ Arc and Aβ wt appeared to be phagocytosed to a larger extent. Scale bar: 20 µm

Article Snippet: The TBS 16K and TBS-T brain extracts were diluted in ELISA incubation buffer (PBS, 0.1% BSA, 0.05% Tween) and incubated over night at 4 °C, followed by detection with 0.25 µg/ml biotinylated anti-TREM2 BAF1729 (R&D), HRP-conjugated streptavidin and K Blue Aqueous TMB substrate and read at 450 nm with a spectrophotometer.

Techniques: Immunostaining, Enzyme-linked Immunosorbent Assay, Sonication, Labeling, Staining

Journal: Acta Neuropathologica Communications

Article Title: Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion

doi: 10.1186/s40478-024-01734-x

Figure Lengend Snippet: Microglial response to Aβ. A Aβ, TREM2 and Iba-1 immunostaining of brain tissue from 18-months-old tg-UppSwe, tg-ArcSwe and tg-Swe mice. Scale bar: 100 µm. ELISA quantification of soluble TREM2 in TBS 16K ( B ) and TBS-T ( C ) brain extracts from 18-months-old wt, tg-UppSwe, tg-ArcSwe and tg-Swe mice. Not significant ( ns ), * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The TBS 16K and TBS-T brain extracts were diluted in ELISA incubation buffer (PBS, 0.1% BSA, 0.05% Tween) and incubated over night at 4 °C, followed by detection with 0.25 µg/ml biotinylated anti-TREM2 BAF1729 (R&D), HRP-conjugated streptavidin and K Blue Aqueous TMB substrate and read at 450 nm with a spectrophotometer.

Techniques: Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica Communications

Article Title: Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion

doi: 10.1186/s40478-024-01734-x

Figure Lengend Snippet: Microglial response to Aβ. A Aβ, TREM2 and Iba-1 immunostaining of brain tissue from 18-months-old tg-UppSwe, tg-ArcSwe and tg-Swe mice. Scale bar: 100 µm. ELISA quantification of soluble TREM2 in TBS 16K ( B ) and TBS-T ( C ) brain extracts from 18-months-old wt, tg-UppSwe, tg-ArcSwe and tg-Swe mice. Not significant ( ns ), * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The TBS 16K and TBS-T brain extracts were diluted in ELISA incubation buffer (PBS, 0.1% BSA, 0.05% Tween) and incubated over night at 4 °C, followed by detection with 0.25 µg/ml biotinylated anti-TREM2 BAF1729 (R&D), HRP-conjugated streptavidin and K Blue Aqueous TMB substrate and read at 450 nm with a spectrophotometer.

Techniques: Immunostaining, Enzyme-linked Immunosorbent Assay