Journal: International Journal of Molecular Sciences

Article Title: Genetic Background and Kinetics Define Wound Bed Extracellular Vesicles in a Mouse Model of Cutaneous Injury

doi: 10.3390/ijms22073551

Figure Lengend Snippet: Antibodies used for analysis of EV surface markers using vesicle flow cytometry.

Article Snippet: CD18 , M18/2 , APC , 130-104-019 , Miltenyi Biotec.

Techniques: Cytometry

Journal: Acta biomaterialia

Article Title: In vivo short-term and long-term host reaction to starch-based scaffolds.

doi: 10.1016/j.actbio.2010.06.020

Figure Lengend Snippet: Fig. 2. Micrographs of the sections of the explanted FB and WS produced starch-based scaffolds after 1 (A–D) and 2 (E–H) weeks of IM implantation in rats. Tissue was immunohistochemically labelled for CD18 and CD3. represents the SPCL scaffold fibres, arrows mark CD18-positive cells and dashed arrows mark CD3-positive cells.

Article Snippet: The histological analysis of crosssection samples was performed after haematoxylin and eosin (HE), and Masson Goldner Trichrome (MGT) staining and immunohistochemistry using a monoclonal mouse anti-human CD3 antibody (Dako, Denmark) with cross-reactivity for rat T lymphocytes, a monoclonal mouse anti-rat CD18 antibody (Serotec, UK) for the integrin b2 chain of recruited leukocytes, and a monoclonal mouse anti-human phosphoinositide 3-Kinase (Pi3K) antibody (BD, Belgium) with cross-reactivity for rat activated and proliferating lymphocytes, following standard protocols.

Techniques: Produced, Starch

Journal: Acta biomaterialia

Article Title: In vivo short-term and long-term host reaction to starch-based scaffolds.

doi: 10.1016/j.actbio.2010.06.020

Figure Lengend Snippet: Fig. 5. Micrographs of the sections of the explanted FB and WS produced starch-based scaffolds after 1 (A–D) and 2 (E–H) weeks of SC implantation in rats. Tissue was immunohistochemically labelled for CD18 and CD3. represents the SPCL scaffold fibres, arrows mark CD18-positive cells and dashed arrows mark CD3-positive cells.

Article Snippet: The histological analysis of crosssection samples was performed after haematoxylin and eosin (HE), and Masson Goldner Trichrome (MGT) staining and immunohistochemistry using a monoclonal mouse anti-human CD3 antibody (Dako, Denmark) with cross-reactivity for rat T lymphocytes, a monoclonal mouse anti-rat CD18 antibody (Serotec, UK) for the integrin b2 chain of recruited leukocytes, and a monoclonal mouse anti-human phosphoinositide 3-Kinase (Pi3K) antibody (BD, Belgium) with cross-reactivity for rat activated and proliferating lymphocytes, following standard protocols.

Techniques: Produced, Starch

Journal: Innate immunity

Article Title: Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets.

doi: 10.1177/1753425915620911

Figure Lengend Snippet: Figure 1. Different activating capacities of CD18-specific mAbs for bovine PMN. Bovine PMN were incubated with three different mouse mAbs specific for CD18. Bound mAbs were cross-linked after addition of goat anti-mouse IgG F(ab0)2 for 10 min. Alternatively, PMN were stimulated with ionomycin for 10 min or left completely untreated. PMN activation was analyzed by flow cytometric assessment of their shape change after drawing a gate to exclude doublets (SSC-A vs. SSC-W). (a) Representative dot plots of one animal showing a shift in the FSC of activated PMN resulting in a higher percentage of cells in the lower right quadrant. (b) The mean forward scatter (FSC A) of PMN was assessed and data of 7 tested animals were presented as mean SEM (* ¼ P < 0.05 in com- parison to HBSS control).

Article Snippet: MAb clones tested were: mouse anti-dog CD18 (clone CA1.4E9; AbD Serotec) and mouse anti-bovine CD18 (clone HuH82A and BAQ30A; Kingfisher, USA).

Techniques: Incubation, Activation Assay, Control

Journal: Innate immunity

Article Title: Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets.

doi: 10.1177/1753425915620911

Figure Lengend Snippet: Figure 2. Analysis of PMN degranulation products. Bovine PMN were pretreated with mouse mAb against CD18 and were then subjected to CD18 cross-linking through addition of goat anti-mouse F(ab0)2 for 10 min. Alternatively, PMN were stimulated with ionomycin for 10 min. The release of PMN secretory vesicles was analyzed flow cytometrically by determining CD16 expression (a) on activated neutrophils (n ¼ 5 animals) after labeling the cells with a murine mAb specific for CD16.The enzymatic activity of alkaline phosphatase (b) was determined in the supernatant of degranulated PMN by colorimetric assays (n ¼ 6 animals, optical densities, OD). Bovine lactoferrin release (c) in the supernatants of degranulated PMN (n ¼ 6 animals) was measured by ELISA. Myeloperoxidase enzymatic activity (d) was determined in the supernatant of degranulated PMN by colorimetric assays (n ¼ 6 animals, optical densities, OD). All data are shown as mean SEM (* ¼ P < 0.05 in comparison to HBSS control).

Article Snippet: MAb clones tested were: mouse anti-dog CD18 (clone CA1.4E9; AbD Serotec) and mouse anti-bovine CD18 (clone HuH82A and BAQ30A; Kingfisher, USA).

Techniques: Expressing, Labeling, Activity Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Innate immunity

Article Title: Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets.

doi: 10.1177/1753425915620911

Figure Lengend Snippet: Figure 4. Flow cytometric analysis of adhesion molecules on bovine monocyte subsets. Bovine MNC were labeled with mouse mAbs specific for CD14 and CD16 to define bovine monocyte subsets. MNC were also labeled with mouse mAbs specific for CD62L (a), CD11b (b), CD31 (c), CD18 (d), CD11a (e) or CD49d (f) and labeled cells were analyzed on the flow cytometer. After dead cell exclusion with propidium iodide, gates were made on cM, intM or ncM subsets in a CD14 versus CD16 dot plot. Mean fluorescence intensities of adhesion molecules were determined for gated bovine cM, intM and ncM (mean SEM, n ¼ 13 animals; * ¼ P < 0.05).

Article Snippet: MAb clones tested were: mouse anti-dog CD18 (clone CA1.4E9; AbD Serotec) and mouse anti-bovine CD18 (clone HuH82A and BAQ30A; Kingfisher, USA).

Techniques: Labeling, Flow Cytometry, Fluorescence

Journal: Innate immunity

Article Title: Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets.

doi: 10.1177/1753425915620911

Figure Lengend Snippet: Figure 5. PMN-DGP-induced modulation of adhesion molecule expression on bovine monocyte subsets. Bovine MNC were incubated without or with bovine PMN-DGP for 2 h. Incubated cells were then labeled with mouse mAbs specific for CD14 and CD16 to define bovine monocyte subsets. MNC were also labeled with mouse mAbs specific for CD62L (a), CD11b (b), CD31 (c), CD18 (d), CD11a (e) or CD49d (f) and labeled cells were analyzed on the flow cytometer. MFI were determined for gated bovine cM, intM and ncM. MFI values of cells in control set ups (MNC in RPMI medium) were set to 100 % (mean SEM; n ¼ 13 animals; * ¼ P < 0.05).

Article Snippet: MAb clones tested were: mouse anti-dog CD18 (clone CA1.4E9; AbD Serotec) and mouse anti-bovine CD18 (clone HuH82A and BAQ30A; Kingfisher, USA).

Techniques: Expressing, Incubation, Labeling, Flow Cytometry, Control

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F Macrophages were treated with LPS (1 µg/mL) for 6, 12, or 24 h with or without PD168393 (PD, 10 µM)) pretreatment for 30 min. A Flow cytometry analysis of EGFR cell surface expression in BMDM. B Percentage of EGFR-positive macrophage is shown. C Mean fluorescence intensity (MFI) is shown ( n = 3). D Flow cytometry analysis of EGFR cell surface expression in RAW264.7. E Percentage of EGFR-positive macrophage is shown. F Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from peritoneal lavage of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and peritoneal macrophages were identified with CD45 + CD11b + F4/80high. G EGFR intensity on the surface of peritoneal macrophage was analyzed by flow cytometry. H Percentage of EGFR-positive peritoneal macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J , K Blood was collected from patients with clinical sepsis or health donors, and blood mononuclear cells were identified with CD14 and CD11c. J EGFR intensity on the surface of Mononuclear cells were analyzed by flow cytometry (20 Healthy donors and 33 Septic patients). K Percentage of EGFR-positive CD14 + CD11c + mononuclear cell is shown. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F BMDMs were treated with LPS (1 μg/mL) for 24 h with or without PD168393 (10 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of IL-1β ( n = 3). B RT-qPCR analysis of mRNA expression of iNOS ( n = 3). C Western blot was used to detect the expression of iNOS. D iNOS expression on the surface of BMDM was analyzed by flow cytometry. E Percentage of iNOS -positive BMDM is shown ( n = 3). F Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G iNOS expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of iNOS-positive alveolar macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J Cluster analysis of differentially expressed metabolites in RAW264.7 measured after treated with LPS (1 μg/mL) for 30 min with or without PD168393 (PD 10 μM) pretreatment for 30 min, when compared with control. Blue and red indicates down-or upregulation, respectively ( n = 3 samples of each condition). K Schematic illustrating the metabolites that are decreased (blue) in PD168393 (10 μM) RAW264.7 cells at 24 h after LPS stimulation. L PD168393 (10 μM) pretreatment RAW264.7 cells exhibited a ~2 -fold decrease in lactate levels compared with LPS group ( n = 3). M Representative western blots of HIF1-a, p-PKM2, PKM2, LDHA expression in RAW264.7 cells. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Control

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h, with or without Erlotinib (20 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of M2-related genes Mcr1 ( n = 3). B RT-qPCR analysis of mRNA expression of M2-related genes Ym1 ( n = 3). C Representative western blot of Arg1. D Flow cytometry analysis showing the level of M2 macrophage-associated markers CD206. E Percentage of CD206-positive RAW264.7 is shown ( n = 3). I Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of CD206-positive alveolar macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J Immunoblot analysis of p-PPARγ (Ser112), t-PPARγ in RAW264.7 cells treated with LPS (1 μg/mL) for 30 min with or without indicated concentration of PD168393 (10 μM) pretreatment for 30 min. K Fluorescence images depicting PPARγ translocation (left panel, scale bar, 50 μm; right panel, scale bar, 5 μm). L – N RAW264.7 cells were treated with LPS (1 μg/mL) for 24 h with or without Erlotinib (10 μM) or Rosiglitazone (Rosi (20 μM) pretreatment. L Cell surface CD206 were analyzed by flow cytometry. M Percentage of CD206-positive RAW264.7 is shown ( n = 3). N Mean fluorescence intensity (MFI) is shown ( n = 3). O – Q Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. O CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. P Percentage of CD206-positive alveolar macrophage is shown ( n = 9). Q Mean fluorescence intensity (MFI) is shown ( n = 9). R – V RAW264.7 cells were treated with LPS (1 μg/mL) for 30 min with or without PD168393 (10 μM) pretreatment for 30 min. R Flow cytometry analysis of JC-1 for the detecting the change of mitochondrial membrane potential (ΔΨm) (left panel, JC-1 aggregates; right panel, JC-1 monomers). S The ratio of JC-1 aggregates /JC-1 monomers was calculated as Δψm. T Total cellular ATP level was detected ( n = 3). U Immunoblot analysis of IRG1, ATP5A, SDHA, Tubulin as a loading control. V Immunoblot analysis of SDHA and IRG1 in Control or IRG1 silenced RAW264.7 cells, Tubulin as a loading control. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Concentration Assay, Translocation Assay, Membrane, Control

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – E Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. A iNOS and CD206 expression on the surface of alveolar macrophage were analyzed by flow cytometry. B Percentage of iNOS-positive alveolar macrophage is shown ( n = 9). C Mean fluorescence intensity (MFI) of iNOS is shown ( n = 9). D Percentage of CD206-positive alveolar macrophage is shown ( n = 9). E Mean fluorescence intensity (MFI) of CD206 is shown ( n = 9). F – J Mice were injected intraperitoneally with LPS (20 mg/kg) or an equal volume of PBS, after pretreatment with Erlotinib (100 mg/kg) for 2 h. F The BALF neutrophils were analyzed by flow cytometry with neutrophils markers (CD11b and Ly6G). G Percentage of CD11b + Ly6G+ positive alveolar macrophage is shown ( n = 9). H Mean fluorescence intensity (MFI) of CD11b + Ly6G+ is shown ( n = 9). I Immune-staining of iNOS and CD206 in Lung sections. J Lung sections were stained with hematoxylin and eosin (H&E). Scale bar, 50 μm (upper panel) and 5 μm (lower panel). The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Expressing, Flow Cytometry, Fluorescence, Injection, Staining