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Image Search Results
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Clustering of all cell subsets from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). b ) Clustering of monocyte/macrophage populations from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). c ) PTPRC and CD14 expression of clustered monocyte/macrophage populations from 3b. d ) Foamy macrophage gene (FABP5, LGALS3) expression of clustered monocyte/macrophage populations from 3b. e ) Inflammatory macrophage gene (IL1B, NLRP3) expression of clustered monocyte/macrophage populations from 3b. f ) TREM2 expression of clustered monocyte/macrophage populations from 3b. g ) Volcano plot of enrichment of genes from samples from either asymptomatic or symptomatic patients. TREM2 in red. DEGs were determined by Wald test with DESeq2.
Article Snippet: The following antibodies were used:
Techniques: Expressing
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for CRISPR knockout screening approach for oxLDL uptake. BV2 macrophages were loaded with CRISPR pooled guide library (Gouda). Cells were made foamy by overnight treatment with soluble cholesterol and then challenged for 4 h with DiI-oxLDL and sorted for DiI high and DiI low cells. Guides were sequenced from sorted populations. b , Confocal micrograph showing BV2 DiI uptake after 4-h incubation with DiI-oxLDL. Representative of two independent experiments. c , CRISPR guide enrichment comparing log-normalized enrichment in DiI high ( x axis) versus DiI low ( y axis). Gray error bands delineate guides with log fold change < 1. d , Selected gene enrichments comparing DiI low versus DiI high . e , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were analyzed for Trem2 expression by flow cytometry ( n = 5 biologically independent replicates per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. f , Bodipy staining for total neutral lipid accumulation was performed by flow cytometry on peritoneal macrophages from WT or Trem2 −/− mice, cultured overnight in media alone or in media with soluble cholesterol ( n = 6 biologically independent replicates for untreated and n = 4 biologically independent replicates for foamy). Data are mean ± s.e.m. Student’s t -test. g , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were treated with DiI-oxLDL for 4 h and assessed for uptake by flow cytometry ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. h , CD36 expression from peritoneal macrophages isolated from WT or Trem2 −/− mice and treated with soluble cholesterol overnight to induce foamy cell formation ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. NS, not significant.
Article Snippet: The following antibodies were used:
Techniques: CRISPR, Knock-Out, Incubation, Isolation, Expressing, Flow Cytometry, Staining, Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Time course analysis of DiI-oxLDL uptake in WT BV2 cells differentiated in media with 20 μg/mL of soluble cholesterol overnight prior to addition of DiI-oxLDL (n = 5 biological replicates/group). Data are mean ± S.E.M. b ) CRISPR guide enrichment by rank-order was plotted against P-value for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. Trem2 in red. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. c ) Two sided P-value vs count and p value vs false discovery rate (FDR) for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. p-values and FDR calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. d ) Top 15 ‘importance index’ genes associated with foamy cell commitment by Trade-seq analysis , were compared for gene rank and enrichment in CRISPR screen. Trem2 highlighted in gray. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. e) CD36 expression (left) and percent CD36 high (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test, P = ** < 0.01. f ) SR-AI expression (left), MFI (middle) and percent SR-AI positive (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test
Article Snippet: The following antibodies were used:
Techniques: CRISPR, Expressing, Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Cranial artery plaques were stained either for CD68 and Trem2 to identify co−expressing foamy macrophages within human plaques (top) or with CD68 and isotype control with secondary antibody (bottom). Representative image from 3 independent samples. b ) Carotid artery endarterectomy samples from three patients stained for isotype control or Trem2 using DAB (3,3′-Diaminobenzidine) immunohistochemical staining. Representative images from 6 independent samples.
Article Snippet: The following antibodies were used:
Techniques: Staining, Expressing, Control, Immunohistochemical staining
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for mixed bone marrow chimera experiment. Ldlr −/− mice were lethally irradiated and rescued by donor bone marrow from (50%) LysM cre R26 tdTomato (WT) and (50%) Trem2 −/− mice. Recipient mice were rested for 8 weeks and then fed an HFD for an additional 8 weeks to induce atherosclerosis. b , Flow cytometry gating of blood immune cells (CD45 + ) after 8-week HFD feeding, showing ratio of monocytes derived from WT (tdTomato + ) and Trem2 −/− progenitors. c , Confocal micrograph of whole-mount aorta showing tdTomato labeling (red) and CD45 (white) staining to define cellular contributions to foamy macrophages. Representative image from two independent experiments. d , Quantification of tdTomato + cells in blood compared to foamy macrophages from whole-mount aorta images ( n = 3 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. e , Foamy FACS was performed on CD64 + CD11b + macrophages isolated from mixed bone marrow chimera aorta. Macrophages were separated into tdTomato + and tdTomato − populations and then assessed for foamy representation by SSC and Bodipy (neutral lipid) staining. f , Flow cytometric overlap between tdTomato + (red) and Trem2 −/− (blue) derived macrophages from digested atherosclerotic aorta. g , Quantification derived from flow cytometric foamy FACS comparing relative contribution to foamy macrophages ( n = 4 mice per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. KO, knockout. MACS, macrophage.
Article Snippet: The following antibodies were used:
Techniques: Irradiation, Flow Cytometry, Derivative Assay, Labeling, Staining, Isolation, Knock-Out
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice (which included Cre − animals CX3CR1 +/+ Trem2 fl/fl Ldlr −/− and Cre + animals CX3CR1 creER/+ Trem2 fl/+ Ldlr −/− ) were fed TAM-HFD for 8 weeks ( b – e ) or 16 weeks ( f – i ). b , After 8 weeks of TAM-HFD, aortas were analyzed by en face analysis for percentage Oil Red O (ORO) staining on the arch ( n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. c , Aortic sinus plaque area measured after ORO staining in 8-week TAM-HFD samples (n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Serum cholesterol levels from 8-week TAM-HFD-fed mice ( n = 11 mice per group for Cntl and n = 8 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Weight data from 8-week TAM-HFD-fed mice ( n = 9 mice pergroup). Data are mean ± s.e.m. f , En face ORO staining of aorta after 16-week TAM-HFD feeding ( n = 12 mice per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. g , Aortic sinus plaque area after 16-week TAM-HFD feeding ( n = 11 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. h , Serum cholesterol after 16-week TAM-HFD feeding ( n = 10 mice per group). Data are mean ± s.e.m. i , Weight of mice after 16-week TAM-HFD feeding ( n = 10 mice per group for Cntl and n = 7 for Trem2 ΔMФ ). Data are mean ± s.e.m.
Article Snippet: The following antibodies were used:
Techniques: Control, Staining
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Trem2 expression and quantification from atherosclerotic aortae (n = 2 mice/group for Cntl and n = 3 for Trem2ΔMФ). Briefly, Cntl or Trem2ΔMФ mice were fed TAM-HFD for 16 weeks then aorta were harvested, digested and flow cytometry was run. Histogram was gated on live, CD45 + CD11b + CD64+ cells. b ) Flow cytometric gating strategy for identifying major blood immune cell populations. c ) Blood immune cell profiling by flow cytometry in indicated mice after 16 weeks TAM-HFD feeding (n = 12 mice/group for Cntl and n = 6 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test. d ) Classical monocyte bead uptake in the blood was measured by flow cytometry 24 hours after i.v. bead injection in indicated strains after 16 weeks TAM-HFD feeding (n = 7 mice/group for Cntl and n = 5 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test.
Article Snippet: The following antibodies were used:
Techniques: Expressing, Flow Cytometry, Injection
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Following the schematic in , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice were treated continuously with TAM-HFD for the indicated times. b , Serum from 8-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 10 mice per group). Data are mean ± s.e.m. c , Serum from 16-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 9 mice per group). Data are mean ± s.e.m. d , Blood immune cells were assessed after 16 weeks of TAM-HFD by flow cytometry ( n = 12 mice per group for Cntl and n = 6 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Monocyte recruitment was assessed by bead labeling and recruitment experiment—images from representative histologic and immunofluorescence images with lipid content (red) and beads (green). Representative image from two independent experiments. f , Quantification of plaque-associated beads that were counted per section for 8-week or 16-week TAM-HFD experiments from experiments in ( n = 7 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test. NS, not significant; ORO, Oil Red O.
Article Snippet: The following antibodies were used:
Techniques: Control, Multiplex Assay, Flow Cytometry, Labeling, Immunofluorescence
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Confocal micrograph showing CD68 staining (green) and DAPI (blue) for macrophage area in Cntl or Trem2-deficent mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. b , Quantification of CD68 + macrophage area per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. c , Quantification of the percentage of plaque that is macrophages (CD68 + ) in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test. d , Confocal micrograph showing Ki67 staining (magenta) and CD68 staining (green) for proliferation in Cntl or Trem2-deficient mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. e , Quantification of Ki67 + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group for 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 6 mice per group for 16-week TAM-HFD Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. f , Confocal micrograph of TUNEL staining (magenta) and CD68 staining (green) for detection of dying cells within atherosclerotic lesions after 16-week TAM-HFD feeding. Representative image from two independent experiments. g , Quantification of TUNEL + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 6 mice per group for Cntl 8-week TAM-HFD, n = 7 mice per group for Trem2 ΔMФ 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 7 mice per group for Trem2 ΔMФ 16-week TAM-HFD). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01.
Article Snippet: The following antibodies were used:
Techniques: Staining, TUNEL Assay
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for intervention study where mice were fed an HFD for 8 weeks and then switched to TAM/HFD for an additional 8 weeks before being killed. b , En face aorta analysis of plaque area after 16 weeks of diet-switch intervention study. Representative image from two independent experiments. c , Quantification of plaque area in aorta and aortic sinus ( n = 8 mice per group for Cntl and n = 9 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Blood immune population analysis after 16-week diet-switch intervention model ( n = 5 mice per group). Data are mean ± s.e.m. e , Total serum cholesterol levels after 16-week diet-switch model ( n = 4 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. f , Quantification of plaque macrophage proliferation analysis by Ki67 + macrophages (CD68 + ) ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. g , Quantification of TUNEL + macrophages (CD68 + ) in plaques after 16-week diet-switch model ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. ORO, Oil Red O.
Article Snippet: The following antibodies were used:
Techniques: TUNEL Assay
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , WT and Trem2 −/− BV2s assessed for Trem2 expression by flow cytometry. WT ( b ) or Trem2 −/− ( c ) BV2 macrophages DEGs from bulk RNA-seq determined by Wald test with DESeq2. d , GSEA plot of cholesterol biosynthesis pathways. ES, enrichment score. e , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− non-foamy BV2 cells. f , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− foamy BV2 cells. e , f , Significant pathways determined using weighted Kolmogorov–Smirnov test. g , WT or Trem2 −/− cell supernatant assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Foamy: 20 μg ml −1 cholesterol; foamy hi : 80 μg ml −1 cholesterol. Data are mean ± s.e.m. Two-tailed ANOVA, *** P < 0.001. h , DiI-oxLDL uptake for WT or Trem2 −/− non-foamy and foamy BV2 macrophages ( n = 6 for non-foamy WT and Trem2 −/− and n = 4 foamy WT and Trem2 −/− biological replicates). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. i , WT or Trem2 −/− non-foamy and foamy BV2 macrophage efferocytosis. Efferocytotic cells were determined by the percent of BV2s that were positive for CTV-labeled splenocytes ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01 and **** P < 0.0001. j , WT or Trem2 −/− non-foamy and foamy BV2 macrophage sXBP1 expression. Tunicamycin was used as a positive control ( n = 6 biological replicates per group). FMO (fluorescence minus one) shows unstained control. Data are mean ± s.e.m. Two-tailed ANOVA, ** P < 0.01. k , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) plus 10 μM PBA. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. l , GSEA plot of cholesterol efflux pathways from RNA-seq. m , GSEA plot of NR1H2 and NR1H3 gene target pathways from RNA-seq. n , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± T0901317 percent cytotoxicity ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. o , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± 10 μM T0901317, assessed for sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. KO, knockout; NS, not significant.
Article Snippet: The following antibodies were used:
Techniques: Expressing, Flow Cytometry, RNA Sequencing, Lactate Dehydrogenase Assay, Two Tailed Test, Labeling, Positive Control, Fluorescence, Control, Knock-Out
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Heat map of nonfoamy macrophages comparing top enriched WT and Trem2−/− genes. b ) Heat map of foamy macrophages comparing top enriched WT and Trem2−/− genes. c ) Normalized enrichment scores for top pathways associated with WT BV2 compared to Trem2−/− BV2 in media alone (nonfoamy) or following foamy differentiation. Significant pathways were determined using Weighted-Kolmogorov-Smirnov (WKS) test. d ) Heat map of nonfoamy and foamy macrophages comparing matrix metalloprotease genes from WT and Trem2−/− BV2. e ) Cytokine supernatant analysis from cultured WT or Trem2−/− cells cultured with either media alone or media with 20 mg/ml soluble cholesterol overnight (n = 3 biological replicates/group). Data are mean ±S.E.M.
Article Snippet: The following antibodies were used:
Techniques: Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) WT or Trem2−/− peritoneal macrophages were differentiated in media control, media with 20 μg/mL or 80 μg/mL soluble cholesterol to induce foamy macrophage formation. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 hours (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = * < 0.05. b ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then cultured with irradiated, cell trace violet (CTV) labeled splenocytes for 2 hours. Percentage of efferocytotic cells were determined by the % of peritoneal macrophages that were positive for CTV labeled splenocytes (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001, ****<0.0001. c ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then assessed for activation of ER stress response by sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001.
Article Snippet: The following antibodies were used:
Techniques: Control, Lactate Dehydrogenase Assay, Two Tailed Test, Cell Culture, Irradiation, Labeling, Activation Assay, Flow Cytometry, Positive Control
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Clustering of all cell subsets from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). b ) Clustering of monocyte/macrophage populations from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). c ) PTPRC and CD14 expression of clustered monocyte/macrophage populations from 3b. d ) Foamy macrophage gene (FABP5, LGALS3) expression of clustered monocyte/macrophage populations from 3b. e ) Inflammatory macrophage gene (IL1B, NLRP3) expression of clustered monocyte/macrophage populations from 3b. f ) TREM2 expression of clustered monocyte/macrophage populations from 3b. g ) Volcano plot of enrichment of genes from samples from either asymptomatic or symptomatic patients. TREM2 in red. DEGs were determined by Wald test with DESeq2.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Expressing
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for CRISPR knockout screening approach for oxLDL uptake. BV2 macrophages were loaded with CRISPR pooled guide library (Gouda). Cells were made foamy by overnight treatment with soluble cholesterol and then challenged for 4 h with DiI-oxLDL and sorted for DiI high and DiI low cells. Guides were sequenced from sorted populations. b , Confocal micrograph showing BV2 DiI uptake after 4-h incubation with DiI-oxLDL. Representative of two independent experiments. c , CRISPR guide enrichment comparing log-normalized enrichment in DiI high ( x axis) versus DiI low ( y axis). Gray error bands delineate guides with log fold change < 1. d , Selected gene enrichments comparing DiI low versus DiI high . e , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were analyzed for Trem2 expression by flow cytometry ( n = 5 biologically independent replicates per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. f , Bodipy staining for total neutral lipid accumulation was performed by flow cytometry on peritoneal macrophages from WT or Trem2 −/− mice, cultured overnight in media alone or in media with soluble cholesterol ( n = 6 biologically independent replicates for untreated and n = 4 biologically independent replicates for foamy). Data are mean ± s.e.m. Student’s t -test. g , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were treated with DiI-oxLDL for 4 h and assessed for uptake by flow cytometry ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. h , CD36 expression from peritoneal macrophages isolated from WT or Trem2 −/− mice and treated with soluble cholesterol overnight to induce foamy cell formation ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. NS, not significant.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: CRISPR, Knock-Out, Incubation, Isolation, Expressing, Flow Cytometry, Staining, Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Time course analysis of DiI-oxLDL uptake in WT BV2 cells differentiated in media with 20 μg/mL of soluble cholesterol overnight prior to addition of DiI-oxLDL (n = 5 biological replicates/group). Data are mean ± S.E.M. b ) CRISPR guide enrichment by rank-order was plotted against P-value for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. Trem2 in red. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. c ) Two sided P-value vs count and p value vs false discovery rate (FDR) for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. p-values and FDR calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. d ) Top 15 ‘importance index’ genes associated with foamy cell commitment by Trade-seq analysis , were compared for gene rank and enrichment in CRISPR screen. Trem2 highlighted in gray. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. e) CD36 expression (left) and percent CD36 high (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test, P = ** < 0.01. f ) SR-AI expression (left), MFI (middle) and percent SR-AI positive (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: CRISPR, Expressing, Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Cranial artery plaques were stained either for CD68 and Trem2 to identify co−expressing foamy macrophages within human plaques (top) or with CD68 and isotype control with secondary antibody (bottom). Representative image from 3 independent samples. b ) Carotid artery endarterectomy samples from three patients stained for isotype control or Trem2 using DAB (3,3′-Diaminobenzidine) immunohistochemical staining. Representative images from 6 independent samples.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Staining, Expressing, Immunohistochemical staining
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for mixed bone marrow chimera experiment. Ldlr −/− mice were lethally irradiated and rescued by donor bone marrow from (50%) LysM cre R26 tdTomato (WT) and (50%) Trem2 −/− mice. Recipient mice were rested for 8 weeks and then fed an HFD for an additional 8 weeks to induce atherosclerosis. b , Flow cytometry gating of blood immune cells (CD45 + ) after 8-week HFD feeding, showing ratio of monocytes derived from WT (tdTomato + ) and Trem2 −/− progenitors. c , Confocal micrograph of whole-mount aorta showing tdTomato labeling (red) and CD45 (white) staining to define cellular contributions to foamy macrophages. Representative image from two independent experiments. d , Quantification of tdTomato + cells in blood compared to foamy macrophages from whole-mount aorta images ( n = 3 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. e , Foamy FACS was performed on CD64 + CD11b + macrophages isolated from mixed bone marrow chimera aorta. Macrophages were separated into tdTomato + and tdTomato − populations and then assessed for foamy representation by SSC and Bodipy (neutral lipid) staining. f , Flow cytometric overlap between tdTomato + (red) and Trem2 −/− (blue) derived macrophages from digested atherosclerotic aorta. g , Quantification derived from flow cytometric foamy FACS comparing relative contribution to foamy macrophages ( n = 4 mice per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. KO, knockout. MACS, macrophage.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Irradiation, Flow Cytometry, Derivative Assay, Labeling, Staining, Isolation, Knock-Out
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice (which included Cre − animals CX3CR1 +/+ Trem2 fl/fl Ldlr −/− and Cre + animals CX3CR1 creER/+ Trem2 fl/+ Ldlr −/− ) were fed TAM-HFD for 8 weeks ( b – e ) or 16 weeks ( f – i ). b , After 8 weeks of TAM-HFD, aortas were analyzed by en face analysis for percentage Oil Red O (ORO) staining on the arch ( n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. c , Aortic sinus plaque area measured after ORO staining in 8-week TAM-HFD samples (n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Serum cholesterol levels from 8-week TAM-HFD-fed mice ( n = 11 mice per group for Cntl and n = 8 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Weight data from 8-week TAM-HFD-fed mice ( n = 9 mice pergroup). Data are mean ± s.e.m. f , En face ORO staining of aorta after 16-week TAM-HFD feeding ( n = 12 mice per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. g , Aortic sinus plaque area after 16-week TAM-HFD feeding ( n = 11 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. h , Serum cholesterol after 16-week TAM-HFD feeding ( n = 10 mice per group). Data are mean ± s.e.m. i , Weight of mice after 16-week TAM-HFD feeding ( n = 10 mice per group for Cntl and n = 7 for Trem2 ΔMФ ). Data are mean ± s.e.m.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Staining
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Trem2 expression and quantification from atherosclerotic aortae (n = 2 mice/group for Cntl and n = 3 for Trem2ΔMФ). Briefly, Cntl or Trem2ΔMФ mice were fed TAM-HFD for 16 weeks then aorta were harvested, digested and flow cytometry was run. Histogram was gated on live, CD45 + CD11b + CD64+ cells. b ) Flow cytometric gating strategy for identifying major blood immune cell populations. c ) Blood immune cell profiling by flow cytometry in indicated mice after 16 weeks TAM-HFD feeding (n = 12 mice/group for Cntl and n = 6 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test. d ) Classical monocyte bead uptake in the blood was measured by flow cytometry 24 hours after i.v. bead injection in indicated strains after 16 weeks TAM-HFD feeding (n = 7 mice/group for Cntl and n = 5 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Expressing, Flow Cytometry, Injection
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Following the schematic in , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice were treated continuously with TAM-HFD for the indicated times. b , Serum from 8-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 10 mice per group). Data are mean ± s.e.m. c , Serum from 16-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 9 mice per group). Data are mean ± s.e.m. d , Blood immune cells were assessed after 16 weeks of TAM-HFD by flow cytometry ( n = 12 mice per group for Cntl and n = 6 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Monocyte recruitment was assessed by bead labeling and recruitment experiment—images from representative histologic and immunofluorescence images with lipid content (red) and beads (green). Representative image from two independent experiments. f , Quantification of plaque-associated beads that were counted per section for 8-week or 16-week TAM-HFD experiments from experiments in ( n = 7 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test. NS, not significant; ORO, Oil Red O.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Multiplex Assay, Flow Cytometry, Labeling, Immunofluorescence
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Confocal micrograph showing CD68 staining (green) and DAPI (blue) for macrophage area in Cntl or Trem2-deficent mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. b , Quantification of CD68 + macrophage area per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. c , Quantification of the percentage of plaque that is macrophages (CD68 + ) in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test. d , Confocal micrograph showing Ki67 staining (magenta) and CD68 staining (green) for proliferation in Cntl or Trem2-deficient mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. e , Quantification of Ki67 + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group for 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 6 mice per group for 16-week TAM-HFD Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. f , Confocal micrograph of TUNEL staining (magenta) and CD68 staining (green) for detection of dying cells within atherosclerotic lesions after 16-week TAM-HFD feeding. Representative image from two independent experiments. g , Quantification of TUNEL + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 6 mice per group for Cntl 8-week TAM-HFD, n = 7 mice per group for Trem2 ΔMФ 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 7 mice per group for Trem2 ΔMФ 16-week TAM-HFD). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Staining, TUNEL Assay
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , Schematic for intervention study where mice were fed an HFD for 8 weeks and then switched to TAM/HFD for an additional 8 weeks before being killed. b , En face aorta analysis of plaque area after 16 weeks of diet-switch intervention study. Representative image from two independent experiments. c , Quantification of plaque area in aorta and aortic sinus ( n = 8 mice per group for Cntl and n = 9 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Blood immune population analysis after 16-week diet-switch intervention model ( n = 5 mice per group). Data are mean ± s.e.m. e , Total serum cholesterol levels after 16-week diet-switch model ( n = 4 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. f , Quantification of plaque macrophage proliferation analysis by Ki67 + macrophages (CD68 + ) ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. g , Quantification of TUNEL + macrophages (CD68 + ) in plaques after 16-week diet-switch model ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. ORO, Oil Red O.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: TUNEL Assay
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a , WT and Trem2 −/− BV2s assessed for Trem2 expression by flow cytometry. WT ( b ) or Trem2 −/− ( c ) BV2 macrophages DEGs from bulk RNA-seq determined by Wald test with DESeq2. d , GSEA plot of cholesterol biosynthesis pathways. ES, enrichment score. e , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− non-foamy BV2 cells. f , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− foamy BV2 cells. e , f , Significant pathways determined using weighted Kolmogorov–Smirnov test. g , WT or Trem2 −/− cell supernatant assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Foamy: 20 μg ml −1 cholesterol; foamy hi : 80 μg ml −1 cholesterol. Data are mean ± s.e.m. Two-tailed ANOVA, *** P < 0.001. h , DiI-oxLDL uptake for WT or Trem2 −/− non-foamy and foamy BV2 macrophages ( n = 6 for non-foamy WT and Trem2 −/− and n = 4 foamy WT and Trem2 −/− biological replicates). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. i , WT or Trem2 −/− non-foamy and foamy BV2 macrophage efferocytosis. Efferocytotic cells were determined by the percent of BV2s that were positive for CTV-labeled splenocytes ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01 and **** P < 0.0001. j , WT or Trem2 −/− non-foamy and foamy BV2 macrophage sXBP1 expression. Tunicamycin was used as a positive control ( n = 6 biological replicates per group). FMO (fluorescence minus one) shows unstained control. Data are mean ± s.e.m. Two-tailed ANOVA, ** P < 0.01. k , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) plus 10 μM PBA. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. l , GSEA plot of cholesterol efflux pathways from RNA-seq. m , GSEA plot of NR1H2 and NR1H3 gene target pathways from RNA-seq. n , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± T0901317 percent cytotoxicity ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. o , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± 10 μM T0901317, assessed for sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. KO, knockout; NS, not significant.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Expressing, Flow Cytometry, RNA Sequencing Assay, Lactate Dehydrogenase Assay, Two Tailed Test, Labeling, Positive Control, Fluorescence, Knock-Out
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) Heat map of nonfoamy macrophages comparing top enriched WT and Trem2−/− genes. b ) Heat map of foamy macrophages comparing top enriched WT and Trem2−/− genes. c ) Normalized enrichment scores for top pathways associated with WT BV2 compared to Trem2−/− BV2 in media alone (nonfoamy) or following foamy differentiation. Significant pathways were determined using Weighted-Kolmogorov-Smirnov (WKS) test. d ) Heat map of nonfoamy and foamy macrophages comparing matrix metalloprotease genes from WT and Trem2−/− BV2. e ) Cytokine supernatant analysis from cultured WT or Trem2−/− cells cultured with either media alone or media with 20 mg/ml soluble cholesterol overnight (n = 3 biological replicates/group). Data are mean ±S.E.M.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Cell Culture
Journal: Nature cardiovascular research
Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis
doi: 10.1038/s44161-023-00354-3
Figure Lengend Snippet: a ) WT or Trem2−/− peritoneal macrophages were differentiated in media control, media with 20 μg/mL or 80 μg/mL soluble cholesterol to induce foamy macrophage formation. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 hours (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = * < 0.05. b ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then cultured with irradiated, cell trace violet (CTV) labeled splenocytes for 2 hours. Percentage of efferocytotic cells were determined by the % of peritoneal macrophages that were positive for CTV labeled splenocytes (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001, ****<0.0001. c ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then assessed for activation of ER stress response by sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001.
Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems);
Techniques: Lactate Dehydrogenase Assay, Two Tailed Test, Cell Culture, Irradiation, Labeling, Activation Assay, Flow Cytometry, Positive Control
Journal: Frontiers in Molecular Biosciences
Article Title: Primary cilia-mediated regulation of microglial secretion in Alzheimer’s disease
doi: 10.3389/fmolb.2023.1250335
Figure Lengend Snippet: Information of antibodies used in figures.
Article Snippet: Primary antibodies, including anti-CD63 (Sicgen, AB0047-200, 1:1,000), anti-CD81 (Novus Biologicals, NB100-65805, 1:1,000), anti-GAPDH (Origene, TA802519, 1:1,000), anti-Ift88 (Proteintech, 60227-1-Ig, 1:1,000), anti-β-Amyloid (D54D2) (Cell Signaling, 8243S, 1:1,000), anti-TMEM119 (Proteintech, 66948-1, 1:1,000) anti-P2RY12 (BioLegend, 848002, 1:1,000), anti-C1Q (Abcam, ab182451, 1:1,000), anti-SPARC (Cell Signaling, 8725, 1:1,000), anti-αTub (Abcam, ab21058, 1:1,000),
Techniques: Staining