Journal: Chemical Engineering Journal

Article Title: Cryogenically 3D printed biomimetic scaffolds containing decellularized small intestinal submucosa and Sr2+/Fe3+ co-substituted hydroxyapatite for bone tissue engineering

doi: 10.1016/j.cej.2021.133459

Figure Lengend Snippet: Fig. 5. In vitro RAW cells polarization modulation after cultured on scaffolds for 3 days. (A) Calcein-AM staining of RAW; (B) RAW cells polarization examined by double marked immunofluorescence of NOS2/Arg-1; (C) RAW polarization quantified by double marked flow cytometry of CD86/CD206; (D) Quantitative CD206 and CD86 expression ratios; (E) RT-qPCR analysis of relative IL6, IL1-β, TNF-α, Arg-1 and IL10 gene expressions; (F) RT-qPCR analysis of relative VEGF, PDGF-BB, TGF-β1 and BMP2 gene expressions. Data represent the mean ± SD. *p < 0.05 versus SIS; #p < 0.05 versus SIS/FeHA.

Article Snippet: Briefly, RAW cells were harvested and incubated with CD86 antibody (553692, BD Pharmingen, USA) under 4 °C for 30 min. Then, cells were washed, resuspended, and incubated with CD206 antibody (FAB25351A, R&D Systems, USA) again to be analyzed using the flow cytometer (Beckman Coulter, CytoFLEX).

Techniques: In Vitro, Cell Culture, Staining, Immunofluorescence, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Chemical Engineering Journal

Article Title: Cryogenically 3D printed biomimetic scaffolds containing decellularized small intestinal submucosa and Sr2+/Fe3+ co-substituted hydroxyapatite for bone tissue engineering

doi: 10.1016/j.cej.2021.133459

Figure Lengend Snippet: Fig. 7. In vivo immunomodulation and vascularization of scaffolds following 7 days subcutaneous implantation. (A) The histological graphs of implanted scaffolds : from the general view of horizontal HE sections to internal sites stained by HE, NOS2, CD206, and CD31 IHC in similar positions; (B). Quantitation of CD206 and NOS2 positive stained areas; (C) Quantitative CD206/NOS2 ratios; (D) Quantitation of new vessels density. Red arrows indicate neo-vessels stained by CD31. Data represent the mean ± SD. *p < 0.05 versus SIS; &p < 0.05 versus SIS/FeHA; #p < 0.05 versus SIS/SrHA.

Article Snippet: Briefly, RAW cells were harvested and incubated with CD86 antibody (553692, BD Pharmingen, USA) under 4 °C for 30 min. Then, cells were washed, resuspended, and incubated with CD206 antibody (FAB25351A, R&D Systems, USA) again to be analyzed using the flow cytometer (Beckman Coulter, CytoFLEX).

Techniques: In Vivo, Staining, Quantitation Assay

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: ZBP1 is mainly expressed in macrophages and alters the ratio of M1 and M2 macrophages in sepsis‐induced myocardial dysfunction. A single‐nucleus mRNA sequencing (snRNA‐seq) was performed in hearts from LPS‐treated WT or Zbp1 −/− mice (for each group, single‐cell suspensions from three hearts were pooled as one sample). (A) UMAP plot of cell clusters in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. CM, cardiomyocyte; EC, endothelial cell; FB, fibroblast; Mac, macrophage; NP, neutrophil. (B,C) UMAP plot and dot plot of Zbp1 expression in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. (D,E) UMAP plot of macrophage in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. M1, M1 macrophage; M2, M2 macrophage. (F–H) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images (F) and quantification of positive cells (G,H) are shown [scale bar = 50 µm, n = 5]. Mean ± SEM; * P < 0.05, *** P < 0.001, ns, no significance. WT, wild‐type; ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; snRNA‐seq, single‐nucleus mRNA sequencing.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Sequencing, Expressing, Marker, Staining, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Myeloid‐specific Zbp1 deficiency protects against sepsis‐induced myocardial dysfunction. (A) Schematic showing experimental design to investigate the impact of myeloid‐specific Zbp1 deficiency on septic myocardial dysfunction (LPS: 10 mg/kg; 6 h). (B) Zbp1 gene knockout efficiency in hearts and bone marrow cells was detected by Western blot. (C–E) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in Zbp1 fl/fl or Zbp1 cko mice. M‐mode analysis of EF (D) and FS (E) before (pre) and 6 h after (post) LPS treatment. [scale bar = 20 µm, n = 7 in each group]. (F–H) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 7 in each group). (I,J) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. LPS, lipopolysaccharide; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Gene Knockout, Western Blot, Staining, Immunohistochemistry, Injection, Marker

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Blocking STAT1 prevents cardiac dysfunction caused by sepsis. (A) Schematic showing experimental design to investigate the impact of STAT1 inhibition on septic myocardial dysfunction (Flu, fludarabine, STAT1 inhibitor, 50 mg/kg/day; Sol, solvent). (B–D) Western blotting detection and quantitative analysis of STAT1 and ZBP1 in myocardial tissues at 6 h after intraperitoneal injection of NS or LPS (10 mg/kg) in solvent or fludarabine‐treated mice ( n = 5 in each group). (E–G) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in solvent or fludarabine‐treated mice. M‐mode analysis of EF (F) and FS (G) before (pre) and 6 h after (post) LPS treatment. [Scale bar = 20 µm, n = 8–9 in each group]. (H–J) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 5 in each group). (k–l) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, *** P < 0.001. ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; STAT1, signal transducer and activator of transcription 1; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Blocking Assay, Inhibition, Solvent, Western Blot, Injection, Staining, Immunohistochemistry, Marker, Binding Assay

Journal: Molecular Metabolism

Article Title: Functional regulation of macrophages by Ces1d-mediated lipid signaling in immunometabolism

doi: 10.1016/j.molmet.2025.102166

Figure Lengend Snippet: Ablation of Ces1d promotes migration and M1-like pro-inflammatory polarization in KO BMDMs . (A) Comparison of migration capability between WT and KO BMDMs. Confluent BMDMs differentiated from WT (top panel) or KO (bottom panel) bone marrow cells were scratched and treated with 0.01% DMSO (Control), LPS, Oleic Acid (OA), or Palmitic Acid (PA). Images were captured by confocal microscopy and analyzed at different time points during the different treatments. Scale bar = 200 μm. (B) BMDM migration rates were compared by analyzing images taken at 0 h and 16 h. Cell density in the scratched areas was measured using ImageJ software. Student's t-test, ∗p < 0.05, ∗∗∗p < 0.0001. (C) Polarization of WT and KO BMDMs was assessed after treatment with DMSO (control), Oleic Acid (OA), Palmitic Acid (PA), and LPS. Immunofluorescence (IF) staining was performed using α-CD11c (red, marker for M1-like state) and α-CD206 (green, marker for M2-like state) antibodies. DAPI was used to stain the nuclei. Scale bar = 250 μm. (D) Statistical analysis of the M1-like (CD11c+) ratio for WT or KO BMDMs treated with DMSO (control), Oleic Acid (OA), Palmitic Acid (PA), and LPS (n = 3/group, 100 cells were counted in each image, Student's t-test, ∗p < 0.05). (E) Statistical analysis of the M2-like ratio (CD206+) for WT or KO BMDMs treated with DMSO (control), Oleic Acid (OA), Palmitic Acid (PA), and LPS (n = 3/group, Student's t-test, ∗p < 0.05).

Article Snippet: Polarization of BMDMs isolated from WT and KO mice were analyzed using goat anti-Ces1d (PA5-47802, Invitrogen), rat anti-mouse CD11c antibody (#ab52632, Abcam), CD206 (R&D System, Cat#MAB2535), and anti-mouse antibody (#MAB2535, R&D Systems).

Techniques: Migration, Comparison, Control, Confocal Microscopy, Software, Immunofluorescence, Staining, Marker

Journal: Molecular Metabolism

Article Title: Functional regulation of macrophages by Ces1d-mediated lipid signaling in immunometabolism

doi: 10.1016/j.molmet.2025.102166

Figure Lengend Snippet: Changed levels of DAG in Ces1d KO BMDMs and mice . (A) DAG levels in lysates of WT and KO BMDMs measured by a ELASA kit (n = 3/group, Student's t-test, ∗p < 0.05). (B) DAG levels in conditional medium collected from WT and KO BMDMs measured by a ELASA kit (n = 3/group, Student's t-test, ∗∗∗p < 0.0001). (C) DAG levels in serum collected from HFD-fed WT and KO mice (n = 4/group, Student's t-test, ∗p < 0.05). (D) DAG levels in liver of HFD-fed WT and KO mice (n = 4/group, Student t-test, ∗p < 0.05). (E) q-PCR analysis of the mRNA levels of TNFα and IL1β in WT and KO BMDMs. Samples were run in triplicate, and statistical significance was assessed using Student's t-test (∗p < 0.05). (F) Western blot analysis of protein levels of TNFα and IL1β in WT and KO BMDMs. (G) The band density of the precursors and cleaved forms of the proteins were analyzed by ImageJ. Samples were run in triplicate, and statistical significance was assessed using Student's t-test (∗p < 0.05). (H) Co-IF staining with α-CD11c (green) and α-CD206 (red) on Raw 267.4 cells treated with conditioned medium collected from WT and KO BMDMs for 24 h (top scale bar: 25 μm; bottom scale bar: 5 μm) (left); Quantification of the pro-inflammatory CD11C staining and CD206 staining (right) by ImageJ (3 random fields were analyzed in both WT and KO slides, Student's t-test, ∗∗p < 0.001; ns: no significance).

Article Snippet: Polarization of BMDMs isolated from WT and KO mice were analyzed using goat anti-Ces1d (PA5-47802, Invitrogen), rat anti-mouse CD11c antibody (#ab52632, Abcam), CD206 (R&D System, Cat#MAB2535), and anti-mouse antibody (#MAB2535, R&D Systems).

Techniques: Western Blot, Staining