ic808p  (R&D Systems)

Journal: Redox Biology

Article Title: The antioxidant enzyme Peroxiredoxin-1 controls stroke-associated microglia against acute ischemic stroke

doi: 10.1016/j.redox.2022.102347

Figure Lengend Snippet: Stroke-associated microglia (SAM) exhibit enhanced antioxidant properties. (A) Subclustering analysis of microglia in the stroke brain. Microglia (10,559 cells) were extracted from CL and IL hemispheres at 24 h and 48 h after tMCAO. (B) UMAP plots showing two microglia clusters from CL (left) and IL (right) hemispheres at 24 h after tMCAO. (C) Stacked bar graph showing numbers of microglia counted from IL and CL hemispheres at 24 h after tMCAO. (D) Projection of microglia on a pseudo-time graph plot displaying the transition from homeostatic microglia (blue) to SAM (yellow). (E) Scatterplot showing scRNA-seq analysis of differentially expressed genes. Red and blue dots indicate genes that were significantly increased and decreased, respectively, in SAM (log2FC>±1.5). (F) Gene ontology analysis of genes found to be differentially expressed in SAM. (G) Violin plots showing the expression of the SAM marker genes, Prdx1, Srxn1, Txn1, Spp1, Mt1 , and Mt2 in microglial subclusters. (H) Prdx1-mediated antioxidant pathways against ROS stress. (I) Expression of DAM marker genes in SAM and homeostatic microglia. Red and blue lines respectively indicate up- and down-regulated marker genes of DAM. Increased or decreased gene expression in SAM versus homeostatic microglia is indicated by red or blue dots, respectively. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were then stained with anti-CD45/APC (BioLegend, 103124), anti-CD11b/BV510 (BioLegend, 101263), anti-Ly6G-FITC (BioLegend, 108417), anti-Ly6c-BV421 (BioLegend, 128014), anti-CD11c-PE/cy7 (BioLegend, 117318), anti-MHCII-APC/cy7 (BioLegend, 107628), anti-CD206-PE (BioLegend, 141732), anti-CD63-PE/cy7 (Biolegend, 143910), anti-Spp1-PE (R&D, IC808P), and anti-Fth1-FITC (biobyt, orb102585) in FACS buffer at 4 °C for 30 min, and washed twice with FACS buffer.

Techniques: Expressing, Marker

Journal: Redox Biology

Article Title: The antioxidant enzyme Peroxiredoxin-1 controls stroke-associated microglia against acute ischemic stroke

doi: 10.1016/j.redox.2022.102347

Figure Lengend Snippet: SAM are potential protective microglia associated with ischemic stroke. (A) Violin plots showing the expression of Spp1 in homeostatic clusters 1 and 2 and SAM. (B) FACS analysis of Spp1 positive microglia population in the IL hemisphere of Prdx1+/+ (n = 5) and Prdx1−/− (n = 5) mice after tMCAO. (C) Representative IHC images for Spp1 positive SAM population in the ipsilateral hemisphere of Prdx1+/+ and Prdx1−/− mice. (D,F) Violin plots showing gene expression levels of the indicated genes in homeostatic clusters 1 and 2 and SAM. (E) Representative IHC images for Cd63 positive SAM population in the ipsilateral hemisphere of Prdx1+/+ mice. (G) Immunoblot analysis of Spp1 and Fth1 in CL and IL hemispheres of Prdx1+/+ and Prdx1−/− mice at 24 h after tMCAO (n = 4). Bar graph showing quantification of blotting images. (H) UMAP plots showing co-expression of Fth1 (red) and Cd63 (green). (I) FACS analysis showing Cd63 and Fth1 expression of Prdx1+/+ IL microglia and Prdx1−/− IL microglia the indicated conditions. Data are presented as Mean Fluorescence Intensity (MFI). (J) FACS analysis of Fth1, Cd63 double positive SAM population proportion and numbers in the IL hemisphere of Prdx1+/+ (n = 5) and Prdx1−/− (n = 5) mice. (K) Representative IHC images for Cd63, Fth1 double positive SAM population in the IL hemisphere of Prdx1+/+ and Prdx1−/− mice. (L) Representative IHC images for Fth1 and c-Caspase3 in infarct regions of Prdx1+/+ and Prdx1−/− microglia (IBA-1). All of mice were used at 24 h after tMCAO. Data were presented mean SEM and analyzed by 2-way ANOVA test or the unpaired two-tailed Student’s t-test, *P<0.05, **P<0.01, ***P<0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were then stained with anti-CD45/APC (BioLegend, 103124), anti-CD11b/BV510 (BioLegend, 101263), anti-Ly6G-FITC (BioLegend, 108417), anti-Ly6c-BV421 (BioLegend, 128014), anti-CD11c-PE/cy7 (BioLegend, 117318), anti-MHCII-APC/cy7 (BioLegend, 107628), anti-CD206-PE (BioLegend, 141732), anti-CD63-PE/cy7 (Biolegend, 143910), anti-Spp1-PE (R&D, IC808P), and anti-Fth1-FITC (biobyt, orb102585) in FACS buffer at 4 °C for 30 min, and washed twice with FACS buffer.

Techniques: Expressing, Western Blot, Fluorescence, Two Tailed Test

Journal: Glia

Article Title: Myeloid‐derived suppressor cells support remyelination in a murine model of multiple sclerosis by promoting oligodendrocyte precursor cell survival, proliferation, and differentiation

doi: 10.1002/glia.23936

Figure Lengend Snippet: Osteopontin is secreted by myeloid‐derived suppressor cells (MDSCs) in vitro and in vivo during experimental autoimmune encephalomyelitis (EAE). (a) Representative images of the dot blots of MDSC conditioned medium (MDSC‐CM) versus the vehicle media obtained from the cytokine and angiogenesis/growth factor arrays. (b) Histogram showing the comparative levels of the different candidate factors. (c) Representative image of the spinal cord of EAE mice labeled for Arg‐I (MDSCs) and osteopontin (OPN), showing that OPN expression is mainly circumscribed to the infiltrated area. (d–f) Higher magnifications of the area framed in (c) (dotted line); arrowheads point to OPN presence (green dots) within MDSCs. Scale bar represents 20 μm in (c) and 12 μm in (d–f)

Article Snippet: According to the proteomic analysis of the composition of MDSC‐CM, to remove the soluble osteopontin we first incubated MDSC‐CM with the anti‐mouse Osteopontin (2 μg/ml; R&D System) antibody for 30 min at 4°C, and then with μMACS Protein G MicroBeads (100 μl/2–4 μg of affinity‐purified polyclonal antibody; Miltenyi) for another 30 min at 4°C and protected from light.

Techniques: Derivative Assay, In Vitro, In Vivo, Labeling, Expressing

Journal: Glia

Article Title: Myeloid‐derived suppressor cells support remyelination in a murine model of multiple sclerosis by promoting oligodendrocyte precursor cell survival, proliferation, and differentiation

doi: 10.1002/glia.23936

Figure Lengend Snippet: Osteopontin is responsible of the oligodendrocyte precursor cell (OPC) survival due to myeloid‐derived suppressor cell (MDSC) secretome. (a–c) Isolated OPCs in culture in the different experimental conditions, immunostained with Olig2 (red fluorescence), and TUNEL to detect apoptotic cells (green fluorescence). (d) The better survival observed in MDSC conditioned medium (MDSC‐CM) came back to control levels when osteopontin was eliminated from that (MDSC‐CM‐OPN condition). Arrows indicate double‐labeled cells. Scale bar represents 53 μm in (a–c). Results of one‐way analysis of variance (ANOVA) with post hoc Tukey test ( n = 4) are represented as: * p < .05

Article Snippet: According to the proteomic analysis of the composition of MDSC‐CM, to remove the soluble osteopontin we first incubated MDSC‐CM with the anti‐mouse Osteopontin (2 μg/ml; R&D System) antibody for 30 min at 4°C, and then with μMACS Protein G MicroBeads (100 μl/2–4 μg of affinity‐purified polyclonal antibody; Miltenyi) for another 30 min at 4°C and protected from light.

Techniques: Derivative Assay, Isolation, Fluorescence, TUNEL Assay, Labeling

Journal: Glia

Article Title: Myeloid‐derived suppressor cells support remyelination in a murine model of multiple sclerosis by promoting oligodendrocyte precursor cell survival, proliferation, and differentiation

doi: 10.1002/glia.23936

Figure Lengend Snippet: Osteopontin is the main effector of myeloid‐derived suppressor cell (MDSC) conditioned medium in promoting oligodendrocyte precursor cell (OPC) proliferation. (a–c) Images of A2B5 + cells in culture in different conditions, immunostained with Olig2 (red fluorescence) and showing BrdU (green fluorescence) incorporation. The increase of OPC proliferation observed with MDSC conditioned medium (MDSC‐CM) came back to control levels when osteopontin was eliminated from that (MDSC‐CM‐OPN condition; d). Arrows indicate double‐labeled cells. Scale bar represents 53 μm in (a–c). Results of multicomparison with post hoc Tukey test ( n = 4) are represented as: * p < .05 and ** p < .01

Article Snippet: According to the proteomic analysis of the composition of MDSC‐CM, to remove the soluble osteopontin we first incubated MDSC‐CM with the anti‐mouse Osteopontin (2 μg/ml; R&D System) antibody for 30 min at 4°C, and then with μMACS Protein G MicroBeads (100 μl/2–4 μg of affinity‐purified polyclonal antibody; Miltenyi) for another 30 min at 4°C and protected from light.

Techniques: Derivative Assay, Fluorescence, Labeling

Journal: Glia

Article Title: Myeloid‐derived suppressor cells support remyelination in a murine model of multiple sclerosis by promoting oligodendrocyte precursor cell survival, proliferation, and differentiation

doi: 10.1002/glia.23936

Figure Lengend Snippet: Oligodendrocyte precursor cell (OPC) differentiation is enhanced by osteopontin secreted by myeloid‐derived suppressor cells (MDSCs). (a–c) Representative images of the culture of OPCs in differentiating conditions, where Olig2 (red fluorescence) stains the entire oligodendroglial lineage and mature oligodendrocytes are labeled with myelin basic protein (MBP) (green fluorescence). The arrows show individual mature OPCs (Olig2 + /MBP + double stained cells). (d) The graph shows a significant decrease in the MBP area when osteopontin is removed when compared to the MDSC‐CM condition. Scale bar represents 53 μm in (a–c). Results of one‐way analysis of variance (ANOVA) and Tukey's post hoc tests are represented as: * p < .05 and *** p < .001

Article Snippet: According to the proteomic analysis of the composition of MDSC‐CM, to remove the soluble osteopontin we first incubated MDSC‐CM with the anti‐mouse Osteopontin (2 μg/ml; R&D System) antibody for 30 min at 4°C, and then with μMACS Protein G MicroBeads (100 μl/2–4 μg of affinity‐purified polyclonal antibody; Miltenyi) for another 30 min at 4°C and protected from light.

Techniques: Derivative Assay, Fluorescence, Labeling, Staining

Journal: Brain, behavior, and immunity

Article Title: A Novel Role for Osteopontin in Macrophage-Mediated Amyloid-β Clearance in Alzheimer’s Models

doi: 10.1016/j.bbi.2017.08.019

Figure Lengend Snippet: (A) Quantification scheme for coronal brain sections. Representative Nissl-stained image of mouse brain at Bregma −2.65 mm. Scale bar: 1mm. Cingulate cortex (CC), hippocampus (HC) and entorhinal cortex (EC) were included in subsequent quantitative analyses. (B) Representative fluorescent micrographs of brains from ADtg and age-matched WT mice, immunolabeled for anti-OPN (red), anti-human Aβ (6E10; green), and nuclei (DAPI, blue). OPN immunostaining was detected within and around Aβ plaques in ADtg mice in all AD-associated brain regions (HC, CC and EC). In WT animals, no Aβ or OPN immunolabeling was detected. (C) Photomicrographs of brain sections (HC and EC) from ADtg mice show OPN immunohistochemistry by labeling with horseradish peroxidase (HRP) -conjugated secondary antibody. OPN is abundant in layers II / III of EC and often forms plaque-like structures. (D) Representative fluorescent micrographs of ADtg mouse brain sections (CC and EC) display immunostaining for OPN (red) and neuronal markers, Tuj1 or NeuN, or the astrocyte marker, GFAP. (E) OPN (red) was expressed by a subset of Iba1+ cells (green) in ADtg mice but not in WT-mice. Scale bars: 100 µm, inserts: 10 µm. (F) Quantitative ELISA analysis of OPN levels in brain lysate from ADtg and WT littermate groups at 13 and 24 months of age (equal numbers of females and males). n = 5–6 mice per group. Fold changes indicated in red. Group means, SEMs and individual data points are shown. **p<0.01, ****p<0.0001, by one-way ANOVA and Tukey’s multiple comparisons post test.

Article Snippet: Samples were centrifuged and cells were stained for intracellular markers with the following antibody: PE-conjugated anti-mouse OPN (#IC808P, R&D).

Techniques: Staining, Immunolabeling, Immunostaining, Immunohistochemistry, Labeling, Marker, Enzyme-linked Immunosorbent Assay

Journal: Brain, behavior, and immunity

Article Title: A Novel Role for Osteopontin in Macrophage-Mediated Amyloid-β Clearance in Alzheimer’s Models

doi: 10.1016/j.bbi.2017.08.019

Figure Lengend Snippet: (A) Schematic illustration of in vitro studies: BM was isolated from WT mice (8- to 12-weeksold) and cultured for 6 or 7 days in MCSF-enriched media to differentiate into macrophages (MΦBM). On day 6, cells underwent overnight treatment with GA, siRNA or minocycline, except for untreated control cells (labeled ‘C’). On day 7, fibrillar Aβ (fAβ40 or fAβ42) was added in a subset of experiments, followed by phagocytosis assays. Brefeldin A (BFA) treatment was performed 3 hours prior to phagocytosis. (B) Constitutive secretion of OPN by MΦBM during a 24 hour period. Primary MΦBM media were collected after 15 min, and then after 1, 4, 6, and 24 hours. OPN protein levels were measured at each time point (ELISA; n=3 wells/time point, 1×106 cells/well, in triplicates). (C–E) Intracellular OPN expression in MΦBM. Scale bars = 10µm. Representative fluorescent micrographs of MΦBM immunostained for OPN, (C) early endosomal antigen (EEA1) marker, (D) late endosome-lysosomal marker Ras-related protein (Rab7), or (E) Golgi marker 58K protein, and nuclei (DAPI). The merged images demonstrate subcellular OPN expression within vesicles, predominantly confined to the trans-Golgi network (yellow punctate signal). (F–J) Upregulation of OPN in MΦBM by GA treatment. (F–G) Representative fluorescent micrographs demonstrate the effect of GA treatment on MΦBM expression of OPN in the absence of Aβ, either (F) without BFA treatment or (G) with BFA pre-treatment. MΦBM were immunostained for OPN (red), which was more highly expressed following GA treatment. (F: insert) Subcellular OPN within transport vesicles in MΦBM. (G) Round-shaped MΦBM after BFA inhibition of OPN secretion. Scale bars: 20 µm, insert scale 5 µm. (H) Quantitative ICC of OPN-immunoreactive area revealed a significant upregulation of OPN in MΦBM following GA treatment, with or without BFA inhibition. Means of individual cell fluorescent areas are indicated (n=5–9 images/well, average 100 cells/image, and n = 3–5 wells/treatment group). (I) Western blot image of cell lysates from above-mentioned experimental groups. (J) Corresponding densitometry results of OPN levels in Western blots (normalized to β-actin levels; n=3 wells/group, 1×106 cells/well; repeated experiments). Group means, SEMs and individual data points are shown. Fold increases in mean values compared with controls indicated in red. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by one-way ANOVA and Dunnett's multiple comparisons post-tests.

Article Snippet: Samples were centrifuged and cells were stained for intracellular markers with the following antibody: PE-conjugated anti-mouse OPN (#IC808P, R&D).

Techniques: In Vitro, Isolation, Cell Culture, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Marker, Inhibition, Western Blot

Journal: Brain, behavior, and immunity

Article Title: A Novel Role for Osteopontin in Macrophage-Mediated Amyloid-β Clearance in Alzheimer’s Models

doi: 10.1016/j.bbi.2017.08.019

Figure Lengend Snippet: (A–H) Representative fluorescent micrographs and quantitative analyses of OPN expression and Aβ uptake in CD68+ MΦBM, in primary cultures pre-treated with GA for 24 hours and stimulated with fibrillar (f)Aβ40 or fAβ42 for 30 min. Scale bars: 10µm. (A) Higher magnification Z-stack image shows intracellular uptake of 6E10+-Aβ along with subcellular OPN expression patterns in untreated MΦBM. (B–C) Increased OPN expression and enhanced cellular uptake of fAβ42 was detected following GA treatment. (D–E) Representative images display phagocytosis of fAβ40 vs. fAβ42 by untreated MΦBM. (F-H) Quantitative ICC analysis of average OPN expression (F), intracellular fAβ40 (G), and fAβ42 (H) areas per MΦBM in GA-treated vs. untreated MΦBM. Along with increased OPN expression, MΦBM pre-treated with GA exhibit enhanced uptake of Aβ fibrils. (I) Representative micrographs of OPN-silenced MΦBM (via siRNAOPN knockdown) vs. untreated cells reveal reduced fAβ42 phagocytosis. Scale bar: 10µm. (J–K) Quantitative ICC revealed that silenced expression of OPN via siRNAOPN leads to impaired fAβ42 phagocytosis in MΦBM, regardless of GA treatment. Negative control scrambled siRNANeg affected neither OPN expression nor fAβ phagocytosis. (L–M) Additional experiment utilizing MΦBM isolated from OPN knockout (KO) mice vs. WT mice (controls, C). Quantitative ICC analysis of intracellular fAβ40 and fAβ42 uptake in control MΦBM, OPN-deficient MΦBM (KO), and GA-treated OPN-deficient MΦBM (KO+GA). OPN-deficient MΦBM exhibit impaired fAβ phagocytosis, partially restored by supplementation of human recombinant (r)OPN. Means of individual cell fluorescent areas indicated (average of n=5–10 images per well, with ~100 cells/image, n = 4–6 wells/treatment group). Group means, SEMs and individual data points are shown. Fold increases in mean values compared with controls indicated in red. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by one-way ANOVA and Tukey's multiple comparisons post-tests or two-tail unpaired Student’s t-test.

Article Snippet: Samples were centrifuged and cells were stained for intracellular markers with the following antibody: PE-conjugated anti-mouse OPN (#IC808P, R&D).

Techniques: Expressing, Negative Control, Isolation, Knock-Out, Recombinant