il 10  (Hycult Biotech)

Journal: Scientific Reports

Article Title: Dual role of YM1+ M2 macrophages in allergic lung inflammation

doi: 10.1038/s41598-018-23269-7

Figure Lengend Snippet: Th2-related and anti-inflammatory cytokines in lungs of healthy mice, vehicle-treated HDM-exposed mice and HDM-exposed mice treated with cynaropicrin during and after induction of allergic lung inflammation. Data are presented as mean ± SEM. *Represents the comparison of healthy versus HDM+ vehicle-treated mice, while #represents the comparison of HDM+ vehicle versus HDM+ cynaropicrin-treated mice.

Article Snippet: YM1+ macrophages were identified by a dual stain using Mac3 and YM1 (anti-mECF-L diluted 1:50, R&D Systems, Oxon, UK, cat# AF2446), and IL-10+ macrophages were identified by a dual stain using Mac3 and IL-10 (anti-IL-10 diluted 1:25, Hycult, Uden, The Netherlands, cat# HP9016).

Techniques: Inhibition, Activation Assay

Journal: Scientific Reports

Article Title: Dual role of YM1+ M2 macrophages in allergic lung inflammation

doi: 10.1038/s41598-018-23269-7

Figure Lengend Snippet: Cynaropicrin treatment changes the balance in macrophage phenotypes. The number of IRF5 + Mac3 + macrophages ( A and B ) and IL-10 + Mac3 + macrophages ( C and D ) in lungs of healthy mice (n = 8), vehicle-treated HDM-exposed mice (n = 16) and HDM-exposed mice treated with cynaropicrin (n = 8) during (left) and after (right) induction of allergic lung inflammation. Representative pictures of the IRF5 + Mac3 + and IL-10 + Mac3 + double-stainings are shown on the right. Nuclear counter staining was not used to maximize visibility of double-positive cells (magnification 200x). Inserts are close up magnifications (magnification 400x) of double-positive cells. Significance was tested using a one-way ANOVA followed by Sidak’s multiple comparisons test comparing healthy vs. HDM+ vehicle and HDM+ vehicle vs. HDM+ cynaropicrin.

Article Snippet: YM1+ macrophages were identified by a dual stain using Mac3 and YM1 (anti-mECF-L diluted 1:50, R&D Systems, Oxon, UK, cat# AF2446), and IL-10+ macrophages were identified by a dual stain using Mac3 and IL-10 (anti-IL-10 diluted 1:25, Hycult, Uden, The Netherlands, cat# HP9016).

Techniques: Staining

Journal: Scientific Reports

Article Title: HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

doi: 10.1038/srep45047

Figure Lengend Snippet: After 20 h incubation with MS-275 (1 μM) followed by 1 h LPS/IFNγ stimulation, RAW264.7 macrophages were prepared for immunofluorescence microscopy ( A ). Green signal represents NF-κB p65 protein, while blue signal visualizes the Hoechst-stained nuclei. MS-275 enhanced the translocation of NF-κB p65 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells, while SAHA (0.41 μM) did not affect the nuclear translocation of NF-κB p65. The presented data are representative images of 3 independent experiments, original magnification 400x. All images were taken under identical instrumental conditions. The effect of MS-275 on NF-κB p65 translocation in LPS/IFNγ-stimulated RAW264.7 macrophages was also analyzed by immunoblotting cell fractions (the blots were cropped; full length blots are shown in ) ( B ) and quantified by densitometry ( C ). As controls for the fractionation PARP-1 (nucleus) and β-actin (cytosol) were used. Data are presented as mean ± SD expressed as fold change compared to control (LPS/IFNγ-treated) cells of 4–5 independent experiments. * p < 0.05 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells. Effects of MS-275 on total NF-κB p65 protein expression in LPS/IFNγ-stimulated RAW264.7 macrophages were analyzed by Western blot (dotted line indicates crop within the same blot; full length blots are shown in ) ( D ) and quantified by densitometry ( E ). Protein levels were normalized against β-actin and control (LPS/IFNγ-treated) cells were set at 100%. Data are presented as mean ± SD of 4 independent experiments and a representative blot is shown in ( D ). ** p < 0.01 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells. qChIP was performed on LPS/IFNγ-stimulated RAW264.7 macrophages treated with MS-275 or vehicle (inhibitor solvent) for 20 h with antibodies against NF-κB p65 ( F ). Enrichment for IL10 regions −48 bp and −220 bp relative to the transcription start site was analyzed by qPCR. Data are presented as mean ± SEM of 2–3 independent experiments and represent fold enrichment compared to normal rabbit IgG (rIgG). * p < 0.05; ** p < 0.01 compared to rIgG.

Article Snippet: Next, sections were incubated overnight at 4 °C with 10 μg/mL anti-human IL-10 rabbit IgG (#HP9016, Hycult Biotech, Uden, The Netherlands).

Techniques: Incubation, Immunofluorescence, Microscopy, Staining, Translocation Assay, Western Blot, Fractionation, Expressing

Journal: Scientific Reports

Article Title: HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

doi: 10.1038/srep45047

Figure Lengend Snippet: MS-275 restored IL10 mRNA levels of cigarette smoke-exposed mice to mRNA levels of air-exposed mice ( A ). Data are presented as means ± SD; n = 4–5 animals per group. * p < 0.05 compared to vehicle (smoke and inhibitor solvent-treated) mice. Numbers of CD68 + and CD68 + /IL-10 + macrophages in the lungs were determined by immunohistochemistry ( B ). Images were taken with identical instrumental conditions and representative images of 4–5 independent mice per group are shown (original magnification 200x). Immunohistochemical stainings were quantified manually and presented as number/mm 2 lung tissue ( C ). Scatter plots showing an increase in CD68 + macrophages upon cigarette smoke exposure. CD68 + /IL-10 + macrophages remained unaffected. MS-275 did not affect the influx of CD68 + or CD68 + /IL-10 + macrophages. Data are presented as mean ± SD; n = 4–5 animals per group. * p < 0.05 compared to air-exposed animals.

Article Snippet: Next, sections were incubated overnight at 4 °C with 10 μg/mL anti-human IL-10 rabbit IgG (#HP9016, Hycult Biotech, Uden, The Netherlands).

Techniques: Immunohistochemistry, Immunohistochemical staining