Journal: BMC Immunology

Article Title: Differential polarization and the expression of efferocytosis receptor MerTK on M1 and M2 macrophages isolated from coronary artery disease patients

doi: 10.1186/s12865-021-00410-2

Figure Lengend Snippet: Cell surface differentiation marker characterization of polarized macrophages. M1 macrophages were polarized using indicated stimuli: GMCSF (20 ng/ml) for 5 days followed by LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 2 days. M2 macrophages were polarized with: MCSF (10 ng/ml) for 5 days, IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 2 days. Polarized macrophages were stained with antibodies against the stated cell surface molecules and fluorescence was measured by flow cytometry. MFI values a were obtained using FlowJo software version 10.7.1 and histograms b from one representative experiment are shown. Bar graph represents mean ± SD, * p < 0.05, ** p < 0.005 with n = 4 for no apparent CAD, n = 3 for non-obstructive CAD and n = 7 for obstructive CAD

Article Snippet: The adherent monocytes were cultured for 5 days in RPMI 1640 with stable glutamine media supplemented with 10% heat-inactivated fetal bovine serum (Capricorn Scientific, Germany), 1% penicillin-streptomycin (Nacalai Tesque, Japan), and 20 ng/ml recombinant GMCSF (Miltenyi Biotec, Germany) for M1 macrophage or 10 ng/ml recombinant MCSF (Gold Biotechnology, Missouri) to generate M2 macrophages.

Techniques: Marker, Staining, Fluorescence, Flow Cytometry, Software

Journal: Journal of Nanobiotechnology

Article Title: HA-coated collagen nanofibers for urethral regeneration via in situ polarization of M2 macrophages

doi: 10.1186/s12951-021-01000-5

Figure Lengend Snippet: The polarization of macrophages to M2 phenotype is related to the elongated cell shape. A Fluorescence micrographs of Raw 264.7 macrophages immune-stained for arginase-1 (green), iNOS (red), and nuclear counterstain (blue) on cell plate (control), collagen and HA-collagen nanofibrous films. Scale bars: 15 μm. B Representative Western blot of arginase-1, iNOS, and tubulin of control, collagen and HA-collagen nanofibrous films and quantification of average across three separate experiments. Quantified TNF-α C and IL-10 D secretion from macrophages cultured on different nanofibrous scaffolds or culture plates using ELISA assay. n = 3, ** p < 0.01, *** p < 0.001

Article Snippet: The supernatant was collected, centrifuged, and used for ELISA after 24, 48, and 72 h. According to the manufacturer’s protocol, TNF-α and IL-10 were quantified by ELISA Kit (FEK0527, EK0417, Boster).

Techniques: Fluorescence, Staining, Control, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 8. Decreased proliferation and increased chemokine production of epidermal keratinocytes from miR-132–KO mice. (A) H&E staining of the skin from miR-132–KO mice (n = 9) and their WT littermate controls (n = 5) and epidermis thickness were analyzed. Scale bars: 100 μm. (B) Immunostaining of Ki-67 and HB-EGF in skin from WT (n = 9) and KO mice (n = 5). Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67–positive cells was counted. Scale bars: 50 μm. (C) Epidermal keratinocytes were isolated from the skin of WT (n = 3) and KO (n = 3) mice. Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, Il1b, and Hbegf expression levels were analyzed using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Staining, Immunostaining, Isolation, Expressing, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 9. Skin wound healing is impaired in miR-132–KO mice. (A) Representative images of wounds on the backs of miR-132–KO mice (n = 9) and their WT littermate controls (n = 6) on days 0, 2, 4, 6, and 8 after wounding. Wound closures were quantified and are presented as the percentage of the initial wound area size. (B) qRT-PCR was performed to detect Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, and Il1b expression levels in the intact and wound-edge skin of both WT (n = 5) and KO (n = 9) mice 2 days after injury. *P < 0.05 and **P < 0.01 by Student’s t test. (C) H&E staining (left panels) and immunostaining of Gr-1 (mid- dle and right panels) in the wound area in WT and KO mice 2 days after injury. White arrows demarcate wound edges. Scale bars: 50 μm. (D) Chemotaxis of neutrophils isolated from the peripheral blood of WT (n = 5) and KO mice (n = 7) toward the medium containing 8 μM N-formylmethionyl-leucyl-phenyla- lanine (fMLP). Migrating cells were quantified by flow cytometry. Plots show the FSC/SSC of the migrated cells. (E) Immunostaining of Ki-67 and HB-EGF in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67– positive cells was counted. (F) Immunostaining of p-STAT3 and p-ERK in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Scale bars: 50 μm (E and F). *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Staining, Immunostaining, Chemotaxis Assay, Isolation, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 11. Inhibition of miR-132 delays reepithelialization of human ex vivo skin wounds. Human ex vivo skin wounds were treated topically with anti–miR-132 (n = 4) or anti–miR-Ctrl (n = 4) after injury, and miR-132 blocking efficiency was confirmed by qRT-PCR (A). (B) H&E staining of the ex vivo skin wounds 3 days after injury. Black arrows demarcate the initial wound edges, while the red arrows indicate a newly formed epidermis. Scale bars: 200 μm. (C) Immunostaining of Ki-67 in ex vivo skin wounds 3 days after injury. Sections were counterstained with DAPI. The number of Ki-67– positive cells was counted. White arrows demarcate the wound edges. Scale bars: 50 μm. CXCL1, CXCL5, CCL20 (D), and HB-EGF (E) expression levels were detected by qRT-PCR in ex vivo wounds treated with anti–miR-Ctrl (n = 4) or anti–miR-132 (n = 4) 3 days after injury. (F) Schematic summary of the regulation and function of miR-132 during skin wound healing. *P < 0.05 and **P < 0.01 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Blocking Assay, Quantitative RT-PCR, Staining, Immunostaining, Expressing

Journal: Scientific Reports

Article Title: Aspirin inhibits LPS-induced macrophage activation via the NF-κB pathway

doi: 10.1038/s41598-017-10720-4

Figure Lengend Snippet: ASA pre-treatment inhibited the activation of LPS-induced in mouse peritoneal macrophage cells. (A) The result of flow cytometric analysis showed that >90% of the cells were macrophages after separation. (B) Schematic representation showed the timing of ASA and LPS treatment in our experiments. (C) The results of western blot showed that before the LPS treatment (1 µg/ml, 24 hrs), the addition of ASA pre-treatment at 200 µg/ml decreased the protein expression of total iNOS. Full-length gels are presented in Supplementary Figure . (D) The result of PCR showed that 200 µg/ml ASA could significantly decrease the LPS-induced expression of iNOS. We also found ASA below 200 µg/ml concentration could downregulate the expression levels of iNOS induced by LPS. However, 200 µg/ml aspirin downregulated the expression level of iNOS more stablely when compared to the other concentrations. (E) The result of ELISA showed the downregulation of TNF-α after ASA pre-treatment (200 µg/ml). (F) Immunocytochemical staining assays showed the LPS-induced iNOS positive cells ratio signaficantly increased after LPS inducement (77.79% ± 3.22%) compared to the control group (11.26% ± 1.39%), and 200 µg/ml ASA pre-treatment made the LPS-induced iNOS positive cells ratio decrease (12.29% ± 2.36%). Scale bar = 50 µm. All results are representative of at least three independent experiments. Results were expressed as mean ± standard deviation (SD), and statistical significance was shown as N P > 0.05, *P < 0.05 or **P < 0.01.

Article Snippet: The Mouse Peritoneal Macrophage Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Activation Assay, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Control, Standard Deviation