dnase biochemical reagent  (Boehringer Mannheim)

Journal: The Journal of Clinical Investigation

Article Title: TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis

doi: 10.1172/JCI135197

Figure Lengend Snippet: (A) Experimental design used to evaluate the role of Trem2 in NAFLD progression. Liver weights (B) and liver weight/body weight ratios (C) of WT and Trem2–/– mice after being fed HFD for 8 weeks. n = 10 in WT group; n = 13 in Trem2–/– group. Levels of TG (D) and cholesterol (E) in serum and livers from mice after being fed HFD for 8 weeks. n = 6 in WT group; n = 9 in Trem2–/– group. (F) Representative images of H&E- and ORO-stained liver sections. n = 4 in WT group; n = 4-5 in Trem2–/– group. Solid and dashed black arrows indicate hepatocytes with macrovesicular fat (Macroves. fat) or hepatocyte ballooning (Hep. balloon.), respectively. Number of hepatocytes with Macroves. fat or Hep. balloon. and percentage of ORO-positive area per high magnification field (HMF) was determined by ImageJ from 6 fields per section. Scale bar: 100 μm. (G) GO analysis of the identified DEGs between WT and Trem2–/– groups (n = 3 per group). (H) Heatmap showing the expression pattern of the identified DEGs (n = 3 per group). The color key indicates the expression levels. (I) Heatmap displays the upregulated and downregulated lipid species in livers (n = 9 mice per group), P < 0.05. The color key indicates the lipid levels. (J) Representative TEM images of mouse hepatocytes (upper) and hepatocellular mitochondria (bottom). n = 4 in WT group; n = 5 in Trem2–/– group. Solid and dashed white arrows refer to complete and fragmented mitochondria, respectively. Scale bars: 10 μm and 1 μm, respectively. (K) ATP levels in WT and Trem2–/– livers. n = 7 per group. All data are shown as the mean ± SD. The data were analyzed by an unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary hepatocytes were washed by washing buffer containing 0.1 g/L DNase (Sinopharm Chemical Reagent Co., Ltd, 64002860), 0.1 g/L MgSO 2 ·7 H 2 O and 0.1 g/L MgCl 2 ·6 H 2 O and then separated via centrifugation at 110 g for 2 minutes.

Techniques: Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis

doi: 10.1172/JCI135197

Figure Lengend Snippet: (A) Schematic representation of the timing strategy used to evaluate the role of KCs in NAFLD progression. n = 12 in WT CTRL group; n =5 in Trem2–/– CTRL group; n =10 in WT GdCl3 group; n =5 in Trem2–/– GdCl3 group. (B and C) Serum and liver triglyceride or cholesterol levels in response to KCs depletion. (D) Representative images of H&E- and ORO-stained liver sections. H&E reveals tissue composition and macrovesicular fat, and ORO visualizes lipid droplets. Number of hepatocytes with macrovesicular fat and percentage of ORO-positive area per HMF was determined by ImageJ from 6 fields per section. Scale bar: 100 μm. (E) ATP contents in the liver. (F and G) Representative image of ORO-stained hepatocytes after coculture with BMDMs (F). BMDMs from WT or Trem2–/– mice were cocultured with primary hepatocytes isolated from WT or Trem2–/– mice in transwell plates. Cultures were added with PA (0.5 mM) for 24 hours in serum-free conditions. Lipid accumulation in hepatocytes was determined by quantification of ORO-positive area as a percentage of whole image area by ImageJ (G). n = 9 per group. Scale bar: 50 μm. (H) ATP content in hepatocytes was quantified by luciferase assay. n = 9 per group. The data were analyzed by 1-way analysis of variance with Bonferroni corrections for multiple comparisons. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary hepatocytes were washed by washing buffer containing 0.1 g/L DNase (Sinopharm Chemical Reagent Co., Ltd, 64002860), 0.1 g/L MgSO 2 ·7 H 2 O and 0.1 g/L MgCl 2 ·6 H 2 O and then separated via centrifugation at 110 g for 2 minutes.

Techniques: Staining, Isolation, Luciferase

Journal: The Journal of Clinical Investigation

Article Title: TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis

doi: 10.1172/JCI135197

Figure Lengend Snippet: (A) GO analysis of DEGs in KCs from WT or Trem2–/– mice fed HFD for 8 weeks. n = 3 per group. (B) Representative TEM images of Exos from WT and Trem2–/– BMDMs incubated with PA (0.5 mM) for 12 hours. Scale bar: 100 nm. (C) Representative results of nanoparticle tracking analysis demonstrate size distribution of Exos derived from WT and Trem2–/– BMDMs. n = 5 per group. (D) WT primary hepatocytes were incubated with WT or Trem2–/– BMDM-derived Exos (20 μg/mL) and PA (0.5 mM) for 12 hours. Lipid accumulation in hepatocytes was determined by quantification of ORO-positive area as a percentage of whole image area by ImageJ. n = 16 in WT group; n = 19 in Trem2–/– group. Scale bar: 25 μm. (E) Experimental design used to evaluate BMDM-Exos in NAFLD progression. n = 6 in WT-Exos group; n = 5 in Trem2–/–-Exos group. (F) Liver weights and liver weight/body weight ratios of mice with Exos treatment. (G and H) Serum and liver triglyceride levels in response to Exos treatment. (I) Representative images of ORO-stained liver sections from mice treated with Exos from WT or Trem2–/– BMDMs. Scale bar: 100 μm. Percentage of ORO-positive area per HMF were determined by ImageJ from 6 fields per section. (J) ATP content in hepatocytes was quantified by luciferase assay. Data are presented as the mean ± SD. The data were analyzed by an unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary hepatocytes were washed by washing buffer containing 0.1 g/L DNase (Sinopharm Chemical Reagent Co., Ltd, 64002860), 0.1 g/L MgSO 2 ·7 H 2 O and 0.1 g/L MgCl 2 ·6 H 2 O and then separated via centrifugation at 110 g for 2 minutes.

Techniques: Incubation, Derivative Assay, Staining, Luciferase

Journal: The Journal of Clinical Investigation

Article Title: TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis

doi: 10.1172/JCI135197

Figure Lengend Snippet: (A) Heatmap of small RNA transcripts in WT and Trem2–/– KCs after 8-week HFD and sequence alignment of miR-106b-5p with 3′ UTRs of mouse (Mmu), human (Hsa), and rat (Rno) Mfn2. n = 4 for each group. The color key indicates the expression levels. (B and C) WT BMDMs were transfected with either NC or miR-106b-5p mimic and then cocultured with primary WT hepatocytes in a transwell plate for 12 hours with 0.5 mM PA stimulation. Lipid accumulation in hepatocytes was determined by quantification of ORO-positive area as a percentage of whole image area by ImageJ (B), n = 9 per group. Scale bar: 25 μm. ATP content in hepatocytes was quantified by luciferase assay (C), n = 6 per group. (D) Representative Western blot images of Mfn2 expression in BNL CL.2 cells, which were transfected with either NC or miR-106b-5p mimic and then stimulated with 0.5 mM PA for 6 hours. The integrated density of the blots was analyzed by ImageJ. n = 3 per group. Data are presented as the mean ± SD. Data were analyzed by an unpaired, 2-tailed Student’s t test. *P < 0.05, ****P < 0.0001.

Article Snippet: Primary hepatocytes were washed by washing buffer containing 0.1 g/L DNase (Sinopharm Chemical Reagent Co., Ltd, 64002860), 0.1 g/L MgSO 2 ·7 H 2 O and 0.1 g/L MgCl 2 ·6 H 2 O and then separated via centrifugation at 110 g for 2 minutes.

Techniques: Sequencing, Expressing, Transfection, Luciferase, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis

doi: 10.1172/JCI135197

Figure Lengend Snippet: (A) Experimental design used to evaluate the role of elevated TREM2 gene dosage in NAFLD progression and sepsis. (B and C) Body weight (B) and liver weight (C) of BAC-TREM2 and littermate WT control mice after a 10-week HFD. n = 11 in WT group; n = 15 in BAC-TREM2 group. Filled and open symbols refer to male and female, respectively. (D–F) Representative images of H&E-stained and ORO-stained liver sections (D). H&E revealed tissue composition, macrovesicular fat, hepatocyte ballooning, and lobular inflammation. ORO visualized lipid droplets. Number of hepatocytes with macrovesicular fat or hepatocyte ballooning, lobular inflammation score, and percentage of ORO-positive area per HMF was determined by ImageJ from 5 fields per section (E and F). n = 7 in WT group; n = 11 in BAC-TREM2 group. Scale bar: 100 μm. (G and H) Levels of hepatic triglyceride (G) and ATP (H) from BAC-TREM2 and littermate WT mice after a 10-week HFD. n = 7 in WT group; n = 11 in BAC-TREM2 group. (I and J) Organ injury and bacteria burden at 24 hours after CLP. Male BAC-TREM2 and WT mice were fed with HFD for 10 weeks. After dietary intervention, mild polymicrobial sepsis was induced by CLP (24-gauge needle). At 24 hours after CLP, livers, lungs, and blood were collected for H&E staining (I) and bacterial burden test (J), respectively. n = 6 in WT group; n = 5 in BAC-TREM2 group. Scale bar: 100 μm. (K) Hepatic ATP levels were quantified by luciferase assay. n = 6 in WT group; n = 5 in BAC-TREM2 group. Data are represented as the mean ± SD. Significance was determined by an unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary hepatocytes were washed by washing buffer containing 0.1 g/L DNase (Sinopharm Chemical Reagent Co., Ltd, 64002860), 0.1 g/L MgSO 2 ·7 H 2 O and 0.1 g/L MgCl 2 ·6 H 2 O and then separated via centrifugation at 110 g for 2 minutes.

Techniques: Control, Staining, Bacteria, Luciferase