mouse 291 trem2 biotinylated antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of siRNA transfection of iMG. Cells were transfected 24 hours after iMG plating. After an additional 24 hours, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG per hour. Data are normalized to mock transfected control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG at endpoint. Data are normalized to mock transfected control. (E) RNA isolated from iMG 24 hours after transfection was used to quantify the percent CD33 mRNA expression by qPCR. (F) Change in TREM2 secretion in conditioned media from iMG was quantified 24 hours after transfection by MSD. (G) Protein lysates isolated from iMG 24 hours after transfection were used to quantify percent SYK phosphorylation by AlphaLISA. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E-G). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Phospho-proteomics, Comparison

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Protein lysates isolated from iMG 72 hours after transduction were used to quantify percent CD33 protein expression by MSD. Data are normalized to no AAV6 transduction control. (C) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour. Data are normalized to no AAV6 transduction control. (E) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry at endpoint. Data are normalized to no AAV6 transduction control. (F) TREM2 secretion in conditioned media was quantified from iMG 72 hours after transduction by MSD. (G)(left) Percent change in TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction with increasing AAV6-CD33 concentrations by MSD. Data are represented as a percent change relative to dose-matched AAV6-mCherry. (right) TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction of AAV6-CD33 by MSD. (MOI = 250000). (H) Protein lysates isolated from HMC3 cells 48 hours after transduction were used to quantify percent full length TREM2 protein expression by MSD. (MOI = 250000). Data represent mean ± SEM using one-way ANOVA with post-hoc Tukey’s multiple comparisons test (B), with uncorrected Fisher’s LSD (E), with post-hoc Dunnett multiple comparisons test (F, G) and one-tailed unpaired t-test (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots represent three independent experimental replicates (D, G).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Transduction, Incubation, Labeling, Isolation, Expressing, Control, Fluorescence, One-tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of the TREM2-CD33 pathway. CD33 inhibition acts on SYK activation, upstream of microglial activation pathways AKT, ERK, p38, and JNK. (B) Protein lysates isolated from THP-1 monocytes 48 hours after transduction were used to quantify percent SYK phosphorylation by MSD. (MOI = 31250) (C) 48 hours post-transduction, THP-1 cells (MOI = 31250) were treated with increasing concentrations of TREM2-targeting antibody agonist (0-10 μg/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (D) 48-hours post-transduction of increasing concentrations of AAV6-CD33 (MOI = 0-31250), THP-1 cells were treated with a TREM2-targeting antibody agonist (2.5 ug/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (E) Percent AKT and GSK3β phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by MSD and ELISA, respectively. (F) Percent ERK and p38-MAPK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (G) Percent JNK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (H) (left to right) Percent expression of secreted IL10, IL1β, TNFα, and IL6 in conditioned media from HMC3 cells 24 hours after LPS challenge and 48 hours after transduction of AAV6-CD33 was quantified by MSD. Data represent mean ± SEM using one-tailed unpaired t-test (A, E-G), and two-way ANOVA using uncorrected Fisher’s LSD with a single pooled variance (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three and two independent experimental replicates (C, D, respectively).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Inhibition, Activation Assay, Isolation, Transduction, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, One-tailed Test

Journal: Cardiovascular Research

Article Title: Single-cell to pre-clinical evaluation of Trem2 , Folr2 , and Slc7a7 as macrophage-associated biomarkers for atherosclerosis

doi: 10.1093/cvr/cvaf210

Figure Lengend Snippet: Comparative analysis of gene expression and sTREM2 levels in mouse and human samples. ( A ) Venn diagram illustrating the overlap of gene expression profiling results from mouse aorta samples analysed by TRAP-Seq and scRNA-Seq, and human coronary arteries analysed by scRNA-Seq, revealing a subset of genes enriched in macrophages. The analysis identifies 388 macrophage-enriched genes, of which 164 are membrane proteins, 33 are metabolic proteins, and 54 are receptor proteins. Sample sizes: TRAP-Seq n = 6 mice, mouse scRNA-Seq n = 3 mice, and human scRNA-Seq n = 4 patients. ( B ) Detailed lists of identified proteins categorized into metabolic proteins and receptor proteins from A . ( C ) Expression of Trem2 , Slc7a7 and Folr2 in TRAP-Seq and scRNA-Seq of mouse aorta. For TRAP-Seq, values represent the average transcripts per million across IP replicates, while for scRNA-Seq, they correspond to cluster pseudobulk counts per million. Gene expression for each method is scaled per gene from 0 to 1, with 1 representing the maximum observed expression. Sample sizes are as indicated in panel A . ( D ) Quantification of soluble (s)TREM2 in the plasma of mice carrying (+) or not carrying (−) the hypercholesterolemic LDLR −/− ApoB 100/100 mutations. The mice were subjected to either a regular diet (0) or a high-fat diet (HFD) for 1 or 3 months. Data are presented for both 12-month-old and 21-month-old mice, illustrating the impact of diet and age. The Wilcoxon rank sum test was used for comparing groups. Sample size (mice per group) at 12 months was 7 (−; HFD 0), 5 (+; HFD 0), 5 (+; HFD 1), 7 (−; HFD 3) and 4 (+; HFD 3), and at 21 months was 25 (−) and 30 (+). ( E , F ) Normalized protein expression (NPX levels of TREM2 in ( E ) human atherosclerotic tissue and ( F ) plasma from individuals of the BiKE cohort in individuals with asymptomatic (AS) and symptomatic (S) carotid stenosis. NPX was calculated as recommended by Olink and is on a log 2 scale in arbitrary units (one NPX difference corresponds to 2-fold in protein concentration). Each dot represents an individual patient's sTREM2 levels, with 50 patients in each group. Statistical significance was measured using a two-sided Student’s t -test with unequal variance. ( G ) Heatmap of Pearson correlation coefficients between plasma and tissue TREM2 levels (atherosclerotic aortic arterial wall = aorta, liver, and mammary artery = m.artery) in 165 coronary artery disease patients from the STARNET cohort. ( H ) Pearson correlation matrix showing relationships between plasma sTREM2, C-reactive protein, serum cholesterol, low-density lipoprotein cholesterol, and triglycerides in 20 individuals from the BiKE cohort. For both panels, the colour scale represents the Pearson correlation coefficient ( r ), ranging from −1 to +1. Asterisks denote statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The plates were washed five times with washing buffer, followed by a 1-h incubation at RT with a biotinylated mouse anti-TREM2 antibody (BAF1729, 1:3000, R&D Systems).

Techniques: Gene Expression, Membrane, Expressing, Clinical Proteomics, Protein Concentration