recombinant mouse xcl1  (R&D Systems)

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 1 | Expression of XCL1 and XCR1 in periprosthetic tissues. (A–C) Detection of XCR1 in human tissues around loosening hip-implant by the immunofluorescence test. Deparaffinized sections were stained to observe F4/80, CD68, iNOS (green), XCR1 (red), and cell nuclei (blue). Scale bars are 100 µm. Representative images exhibit the sectioned tissues from three patients. (D) Detection of XCL1 in synovial fluid from same patients by Western blotting analysis. (E) Gene expressions of XCL1 and XCR 1 in calvarial bone tissues in a murine osteolysis calvarial model. Results represent the means of relative expression values ± SEM of three mice. *indicates a significant difference as determined by the Student t-test (p ≤0.05).

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Expressing, Staining, Western Blot

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 2 | Administration of XCL1 exaggerates osteolytic lesions in a polyethylene-particles-induced osteolysis model. (A) Representative images for micro-CT of calvariae. The right panel shows quantification of the lytic area on the calvarial bone tissues of mice. Results represent the means ± SEM of seven mice. *represents the significance determined by one-way ANOVA, followed by a Tukey’s multiple-comparison procedure. Arrows indicate osteolytic lesions. (B) Representative images for histological analyses of bone sections stained by H&E and TRAP. Scale bar is 100 µm. Arrows indicate bone lesions. (C) Cell count of inflammatory cells in calvarial bone sections. (D) Quantification of TRAP-stained areas in calvarial bone sections. The results represent the means ± SEM for three mice. *indicates a significant difference, as determined by one-way ANOVA, followed by the Tukey’s multiple-comparison procedure (p ≤0.05).

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Micro-CT, Comparison, Staining, Cell Counting

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 3 | Blockade of XCL1/lymphotactin by neutralizing antibody ameliorates severity of osteolysis triggered by polyethylene-particles in murine model. (A) Quantification of the lytic area on the calvarial bone tissues of mice determined by micro-CT. Left panel shows representative images of calvariae. Results represent the means ± SEM of seven mice. *represents the significance determined by one-way ANOVA, followed by a Tukey’s multiple-comparison procedure. Arrows indicate osteolytic lesions. (B) Histological analyses of bone sections stained by H&E and TRAP. Scale bar is 100 µm. (C) Cell count of inflammatory cells in calvarial bone sections. (D) Quantification of TRAP-stained areas in calvarial bone sections. Arrows indicate bone lesions. The results represent the means ± SEM for four mice. *indicates a significant difference, as determined the Student t-test (p ≤0.05).

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Micro-CT, Comparison, Staining, Cell Counting

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 4 | Sponge-soaked protein-induced murine model. (A) Representative images for micro-CT of calvariae. Arrows indicate osteolytic lesions. (B) Quantification of the lytic area on the calvarial bone tissues of mice determined by micro-CT. Results represent the means ± SEM of six mice. *represents the significance determined by one-way ANOVA, followed by a Tukey’s multiple-comparison procedure. (C) Representative images for histological analyses of bone sections stained by H&E and TRAP. Scale bar is 100 µm. Arrows indicate bone lesions. (D) Quantification of TRAP-stained areas in calvarial bone sections. Results represent the means ± SEM of three mice. *indicates a significant difference, as determined by one-way ANOVA, followed by the Tukey’s multiple-comparison procedure (p ≤0.05). (E) Heat map for the gene expression of inflammatory and osteoclast marker genes in bone tissues. Calvarial bone tissues were harvested for the analysis of gene expressions after the implantation of XCL1-soaked sponges. Scale bar (Log2) represents the means of relative expression values of each target gene after normalizing with the GAPDH ± SEM of three mice. *indicates a significant difference, as determined by the t-test (p ≤0.05).

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Micro-CT, Comparison, Staining, Gene Expression, Marker, Expressing

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 5 | Effect of XCL1 on osteoclast differentiation and bone resorption. (A) Count of TRAP-positive cells in RANKL-stimulated monocytes in the presence or absence of XCL1. Left panel shows representative images for cells stained by TRAP. Results represent the means ± SEM of triplicates. *indicates a significant difference, as determined by the Tukey’s multiple comparisons test (p ≤0.05). (B) Actin ring staining assay for cells in RANKL-stimulated monocytes in the presence or absence of XCL1. (C) Quantification of the bone resorbed areas on dentin slices. Results represent the means ± SEM of values from three dentin slices. Left panel shows representative images for the resorbed areas. Scale bars are 200 µm.

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Staining

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 7 | The summary of the current study. XCL1 promotes cells infiltrate, inflammatory response, and osteoclast differentiation leading to aseptic loosening. Scale bars are indicated on histological images. Blocking of the XCL1 might be a potent therapeutic target for this clinical problem.

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Blocking Assay

Journal: Frontiers in immunology

Article Title: Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.

doi: 10.3389/fimmu.2020.01720

Figure Lengend Snippet: FIGURE 6 | Effect of XCL1 on osteoblast activation and function. (A) Effect of XCL1 on the gene expression of inflammatory and osteoclastogenic factors in differentiated osteoblasts. Results represent the means ± SEM of triplicates and *indicates a significant difference, as determined by the Tukey’s multiple comparisons test (p ≤0.05). (B) Effects of stimulation by recombinant proteins on NFKB, SAPK/JNK, P42/p44-MAPK (Erk1/2), and P38- activities. Human osteoblasts were cultured in a differentiation medium supplemented with either XCL1 or TNFα (positive control) and harvested for the gene expression analysis by qRT-PCR or for protein analysis by western blotting.

Article Snippet: For the XCL1-induced osteolysis model, collagen sponges of the single layer type (PELNAC, Tokyo, Japan) were soaked in 1 or 2 μg of recombinant mouse XCL1 (R&D system) or PBS (n = 6 for each group) and transplanted subcutaneously onto the calvariae for 7 days (13).

Techniques: Activation Assay, Gene Expression, Recombinant, Cell Culture, Positive Control, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet: ( A, B ) Quantification of chemokine mRNA levels by qRT-PCR in OT1 ( A ) or gBT1 ( B ) CTLs exposed to H-2K b /SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or non-cognate unpulsed EL4 tumour cells, as indicated. Data is normalised to universal mouse cDNA (positive control). Data points from four independent experiments; bars indicate mean. ND: not detected. ( C ) Quantification of absolute chemokine concentration detected in non-cognate and cognate OT1 supernatants by ELISA (for CCL1 and CCL9) or CBA (for CCL3, CCL4, CCL5, CXCL10, IFN-γ, and TNF-α). Data points from four independent experiments; red lines indicate mean. ND: not detected. ( D ) Transmigration of OT1 CTLs towards basal medium containing indicated concentrations of recombinant chemokine. Data normalised to basal medium (dashed line). Data points from three independent experiments; bars indicate mean. Error bars represent standard deviation. p- values are displayed when means are significantly different compared to the 1 ng/ml condition. ( E ) Transmigration of OT1 CTLs towards chemokine-containing basal medium in the presence of corresponding neutralising antibodies or isotype (IgG) control antibody. Data points from four independent experiments; bars indicate mean. Error bars represent standard deviation. ( F ) Transmigration of CTLs towards cognate supernatant in the presence of neutralising antibodies or IgG isotype control. Data normalised to basal medium (dashed line). Data points from at least three independent experiments; bars indicate mean. Error bars represent standard deviation. p-values are displayed when means are significantly different to both the negative control and the IgG isotype control conditions. ( G ) FMI of OT1 CTLs adjacent to tumouroids containing non-cognate or cognate tumour cells with pre-embedded tumour-reactive CTLs in the presence of chemokine-neutralising antibodies as indicated. ( H ) Time course of CCL3 and CCL4 concentrations in supernatants from CTLs sorted by FACS from 4 hr conjugations with cognate tumour cells (0 hr: completion of conjugation). Solid line: cumulative concentration. Dotted line: differential concentration per hour (difference of cumulative concentrations at consecutive timepoints divided by number of hours in-between). Error bars and shaded areas: range. ( I ) FMI of OT1 CTLs adjacent to masses containing CTLs only (no tumour cells) sorted from conjugations with cognate or non-cognate tumour cells. Comparisons of transmigration indices in ( C ) to ( E ) were performed using ANOVA and Tukey’s multiple comparisons tests. In ( G ) and ( I ), box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. ns: p>0.05, p-values from two-tailed Wilcoxon signed rank test compared to a theoretical median of 0. Figure 4—figure supplement 1—source data 1. Source data file for .

Article Snippet: Peptide, recombinant protein , Recombinant Mouse XCL1 , R & D systems , Cat. #: 486-LT-025 , (1, 10 and 100 ng/ml).

Techniques: Quantitative RT-PCR, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transmigration Assay, Recombinant, Standard Deviation, Control, Negative Control, Conjugation Assay, Two Tailed Test

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet: ( A ) Transmigration of human T cells towards supernatant from T cells conjugated with IgG-coated beads (control beads), recombinant human CCL3 and CCL4, or supernatant from T cells conjugated with αCD3/CD28-coated beads in the absence or presence of CCR2/CCR5 dual antagonist (Cenicriviroc), relative to transmigration towards basal medium. Bars: mean from four independent experiments (data points). Error bars: SEM. p-values from ANOVA and Tukey’s multiple comparison test. ( B ) Cell trajectories of primary polyclonal human T cells adjacent to a tumouroid containing human T cells stimulated with control or αCD3/CD28 beads. Random representative tracks from dataset of three independent experiments, quantified in ( C ). ( C ) FMI of polyclonal human T cells as per ( B ), also assessed in the presence of Cenicriviroc. Box-whiskers indicate medians and the interquartile range (IQR). Red bars: mean of pooled data from three independent experiments. Data points: mean of each individual experiment. n: number of tracks for each experimental condition. ns: p>0.05, p-value from two-tailed Wilcoxon signed rank test compared to hypothetical median of 0. ( D ) Human polyclonal T cells (green) responding to a tumouroid containing T cells (magenta) stimulated with control (top) or αCD3/CD28 beads (bottom). Insets highlight CTLs infiltrating the tumouroid. Scale bars: 500 μm. ( E, F ) Swarming index and density kymographs quantify human T cell movements with respect to tumouroids in ( D ). r: distance from tumouroid edge. ( G ) Human CAR T cells (green) responding to tumouroids containing additional CAR T cells (magenta) and control fibroblast cells (top) or target glioblastoma cells (bottom). Insets highlight CTLs infiltrating the tumouroid. Scale bars: 500 µm. ( H, I ) Swarming index and density kymographs quantify CAR T cell movements with respect to tumouroids in ( G ). r: distance from tumouroid edge. Figure 6—source data 1. Source data file for .

Article Snippet: Peptide, recombinant protein , Recombinant Mouse XCL1 , R & D systems , Cat. #: 486-LT-025 , (1, 10 and 100 ng/ml).

Techniques: Transmigration Assay, Control, Recombinant, Comparison, Two Tailed Test

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant Mouse XCL1 , R & D systems , Cat. #: 486-LT-025 , (1, 10 and 100 ng/ml).

Techniques: In Vivo, Cell Culture, Transduction, Construct, Sequencing, Produced, Transfection, Expressing, Plasmid Preparation, Clone Assay, Control, Recombinant, In Vitro, Mass Spectrometry, Cell Isolation, Selection, Reverse Transcription, Sandwich ELISA, Cytometry, Software

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 plasma levels rise after running and XCL1 treatment increases the number of neurospheres. ( a ) XCL1 plasma levels measured by an ELISA in standard-housed mice (STD, n = 5 mice) and mice housed for 4 days with a running wheel (RUN, n = 6 mice). * p < 0.05, Student’s t -test. ( b ) qPCR gene expression analysis of lymphotactin receptors reveals that neural precursor cells express Itga9 but not Xcr1 (left), although both Itga9 and Xcr1 are detected in splenic control tissue (right). Uncropped gels are presented in Supplementary Fig. . ( c ) Representative images of a SVZ neurosphere (top) and a DG neurosphere (bottom). Scale bars: 100 μm. ( d ) Neurosphere assays with DG-derived primary cells cultured with XCL1. n = 9 to 10 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Neurosphere assays with SVZ-derived primary cells cultured with XCL1. n = 6 to 9 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Neurosphere assays with XCL1-neutralizing antibodies. n = 3 to 6 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( g ) Size distribution of DG-derived neurospheres cultured with XCL1. n = 7 to 8 independent experiments. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression, Control, Derivative Assay, Cell Culture

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 promotes neuronal differentiation in adherent monolayer and neurosphere cultures. ( a ) Viability assay in adherent NPC cultures with XCL1. n = 5 to 6 independent experiments. ( b ) CFSE proliferation assay in adherent NPC cultures with XCL1. n = 3 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( c ) Motility of adherent monolayer-cultured NPCs determined by semi-automated tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. XCL1: n = 92 cells, Control: n = 104 cells, *** p < 0.001, Student’s t -test. ( d ) Quantifica t ion of β-tubulin + cells in proliferating NPC cultures two days after the addition of XCL1. n = 4 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Representative image of differentiated NPCs in adherent monolayer cultures showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( f ) Quantification of GFAP + and β-tubulin + cells in differentiated adherent monolayer cultures treated with XCL1. n = 4 to 5 independent experiments, * p < 0.05, *** p < 0.001, one-way ANOVA with Dunnett test. ( g ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( h ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures treated with XCL1. n = 5 independent experiments, *** p < 0.001, one-way ANOVA with Dunnett test. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Viability Assay, Proliferation Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 influences the cell cycle progression of NPCs in vitro . ( a ) Representative images of a dividing NPC followed by time-lapse microscopy. Images are 5 min apart. Yellow arrows mark the process of cell division. Scale bar: 10 μm. ( b ) Example of a generation tree of a re-dividing cell obtained from semi-automated cell tracking of NPCs to calculate the mean generation time. ( c ) Generation time of NPCs cultured with and without XCL1 determined by semi-automated cell tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. Control: n = 23 cells, XCL1: n = 26 cells. ( d ) Representative flow cytometry plots of the click-iT EdU proliferation assay. Viable cells were first defined using forward scatter and side scatter (left). Doublets were then excluded from single cell signals by plotting Hoechst-width against Hoechst-area (middle). Finally, to determine the cell cycle phase, the DNA content (Hoechst label) was plotted against the EdU signal (right). ( e ) Percentage of NPCs in S phase. n = 4 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Percentage of NPCs in G2/M phases. n = 4 independent experiments. ( g ) Percentage of NPCs in G1/G0 phases. n = 4 independent experiments. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: In Vitro, Time-lapse Microscopy, Cell Tracking Assay, Cell Culture, Control, Flow Cytometry, Proliferation Assay

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: Neurogenesis in XCL1 KO mice is reduced ex vivo . ( a ) Neurosphere assays with primary DG cells from XCL1 KO mice (−/−) and WT littermates (+/+). n = 6 mice per group, * p < 0.05, paired Student’s t -test. ( b ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( c ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures from XCL1 −/− and +/+ mice. n = 4 mice per group, * p < 0.05, Student’s t -test. ( d ) and ( e ) Neurosphere assays with primary DG cells from XCL1 −/− and +/+ mice in the presence of ( d ) potassium chloride (n = 4 to 5 independent experiments) and ( e ) norepinephrine (n = 6 independent experiments). *** p < 0.001, **** p < 0.0001, Student’s t -test.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Ex Vivo

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 plasma levels rise after running and XCL1 treatment increases the number of neurospheres. ( a ) XCL1 plasma levels measured by an ELISA in standard-housed mice (STD, n = 5 mice) and mice housed for 4 days with a running wheel (RUN, n = 6 mice). * p < 0.05, Student’s t -test. ( b ) qPCR gene expression analysis of lymphotactin receptors reveals that neural precursor cells express Itga9 but not Xcr1 (left), although both Itga9 and Xcr1 are detected in splenic control tissue (right). Uncropped gels are presented in Supplementary Fig. . ( c ) Representative images of a SVZ neurosphere (top) and a DG neurosphere (bottom). Scale bars: 100 μm. ( d ) Neurosphere assays with DG-derived primary cells cultured with XCL1. n = 9 to 10 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Neurosphere assays with SVZ-derived primary cells cultured with XCL1. n = 6 to 9 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Neurosphere assays with XCL1-neutralizing antibodies. n = 3 to 6 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( g ) Size distribution of DG-derived neurospheres cultured with XCL1. n = 7 to 8 independent experiments. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Unless otherwise indicated, NPCs from an 80% confluent culture were seeded at a density of 2 × 10 4 cells/cm 2 into PDL/laminin-coated wells with or without coverslips and cultured in proliferation medium (neural basal medium containing 0.5% B-27 ® supplement (50×), 0.25% penicillin/streptomycin (10,000 U/ml) and 0.25% GlutaMAXTM (100X), 20 ng/ml EGF and 20 ng/ml FGF-2) for 48 h. Recombinant mouse XCL1 protein (R & D Systems) was reconstituted at 100 μg/ml stock solution in 0.1% BSA in PBS.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression, Control, Derivative Assay, Cell Culture

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 promotes neuronal differentiation in adherent monolayer and neurosphere cultures. ( a ) Viability assay in adherent NPC cultures with XCL1. n = 5 to 6 independent experiments. ( b ) CFSE proliferation assay in adherent NPC cultures with XCL1. n = 3 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( c ) Motility of adherent monolayer-cultured NPCs determined by semi-automated tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. XCL1: n = 92 cells, Control: n = 104 cells, *** p < 0.001, Student’s t -test. ( d ) Quantifica t ion of β-tubulin + cells in proliferating NPC cultures two days after the addition of XCL1. n = 4 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Representative image of differentiated NPCs in adherent monolayer cultures showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( f ) Quantification of GFAP + and β-tubulin + cells in differentiated adherent monolayer cultures treated with XCL1. n = 4 to 5 independent experiments, * p < 0.05, *** p < 0.001, one-way ANOVA with Dunnett test. ( g ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( h ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures treated with XCL1. n = 5 independent experiments, *** p < 0.001, one-way ANOVA with Dunnett test. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Unless otherwise indicated, NPCs from an 80% confluent culture were seeded at a density of 2 × 10 4 cells/cm 2 into PDL/laminin-coated wells with or without coverslips and cultured in proliferation medium (neural basal medium containing 0.5% B-27 ® supplement (50×), 0.25% penicillin/streptomycin (10,000 U/ml) and 0.25% GlutaMAXTM (100X), 20 ng/ml EGF and 20 ng/ml FGF-2) for 48 h. Recombinant mouse XCL1 protein (R & D Systems) was reconstituted at 100 μg/ml stock solution in 0.1% BSA in PBS.

Techniques: Viability Assay, Proliferation Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 influences the cell cycle progression of NPCs in vitro . ( a ) Representative images of a dividing NPC followed by time-lapse microscopy. Images are 5 min apart. Yellow arrows mark the process of cell division. Scale bar: 10 μm. ( b ) Example of a generation tree of a re-dividing cell obtained from semi-automated cell tracking of NPCs to calculate the mean generation time. ( c ) Generation time of NPCs cultured with and without XCL1 determined by semi-automated cell tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. Control: n = 23 cells, XCL1: n = 26 cells. ( d ) Representative flow cytometry plots of the click-iT EdU proliferation assay. Viable cells were first defined using forward scatter and side scatter (left). Doublets were then excluded from single cell signals by plotting Hoechst-width against Hoechst-area (middle). Finally, to determine the cell cycle phase, the DNA content (Hoechst label) was plotted against the EdU signal (right). ( e ) Percentage of NPCs in S phase. n = 4 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Percentage of NPCs in G2/M phases. n = 4 independent experiments. ( g ) Percentage of NPCs in G1/G0 phases. n = 4 independent experiments. All data represent the mean ± SEM.

Article Snippet: Unless otherwise indicated, NPCs from an 80% confluent culture were seeded at a density of 2 × 10 4 cells/cm 2 into PDL/laminin-coated wells with or without coverslips and cultured in proliferation medium (neural basal medium containing 0.5% B-27 ® supplement (50×), 0.25% penicillin/streptomycin (10,000 U/ml) and 0.25% GlutaMAXTM (100X), 20 ng/ml EGF and 20 ng/ml FGF-2) for 48 h. Recombinant mouse XCL1 protein (R & D Systems) was reconstituted at 100 μg/ml stock solution in 0.1% BSA in PBS.

Techniques: In Vitro, Time-lapse Microscopy, Cell Tracking Assay, Cell Culture, Control, Flow Cytometry, Proliferation Assay

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: Neurogenesis in XCL1 KO mice is reduced ex vivo . ( a ) Neurosphere assays with primary DG cells from XCL1 KO mice (−/−) and WT littermates (+/+). n = 6 mice per group, * p < 0.05, paired Student’s t -test. ( b ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( c ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures from XCL1 −/− and +/+ mice. n = 4 mice per group, * p < 0.05, Student’s t -test. ( d ) and ( e ) Neurosphere assays with primary DG cells from XCL1 −/− and +/+ mice in the presence of ( d ) potassium chloride (n = 4 to 5 independent experiments) and ( e ) norepinephrine (n = 6 independent experiments). *** p < 0.001, **** p < 0.0001, Student’s t -test.

Article Snippet: Unless otherwise indicated, NPCs from an 80% confluent culture were seeded at a density of 2 × 10 4 cells/cm 2 into PDL/laminin-coated wells with or without coverslips and cultured in proliferation medium (neural basal medium containing 0.5% B-27 ® supplement (50×), 0.25% penicillin/streptomycin (10,000 U/ml) and 0.25% GlutaMAXTM (100X), 20 ng/ml EGF and 20 ng/ml FGF-2) for 48 h. Recombinant mouse XCL1 protein (R & D Systems) was reconstituted at 100 μg/ml stock solution in 0.1% BSA in PBS.

Techniques: Ex Vivo

Journal: Neuroscience

Article Title: A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability

doi: 10.1016/j.neuroscience.2018.03.030

Figure Lengend Snippet: XCL1 increases expression of c-Fos, pERK and pp38 in Vc that is blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). Distribution of immunolabeling for (A) c-Fos, (B) pERK and (C) pp38 in Vc following a 2-hour incubation with drug-free aCSF (control), XCL1, XCL1 + vMIP-II, or vMIP-II. c-Fos, pERK and pp38 labeling is localized to the most superficial layers of Vc and is more pronounced in XCL1-exposed Vc tissue. Incubation of trigeminal brainstem slices with XCL1 (2 h) resulted in an increased activation of c-Fos (D), pERK (E) and pp38 (F) in the superficial layers of Vc. vMIP-II blocked XCL1-induced activation of c-Fos (D), pERK (E) and pp38 (F) in the superficial layers of Vc. Numbers in parenthesis indicate animals used. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (ANOVA with Dunnett’s post-hoc test). Data are expressed as the mean ± SEM. Scale bar = 500 μm.

Article Snippet: Slices were transferred to a holding chamber, where they were submerged in oxygenated aCSF maintained at 35 °C and incubated for 1 h. The slices were then incubated for 2 h with one of the following, diluted in aCSF: mouse recombinant XCL1 (0.1 µM; Sigma–Aldrich, UK); XCL1 antagonist vMIP-II (0.1 µM; R&D Systems, Minneapolis, MN, USA); normal aCSF; or vMIP-II (0.1 µM) plus XCL1 (0.1 µM) prior to the electrophysiological recordings.

Techniques: Expressing, Immunolabeling, Incubation, Labeling, Activation Assay

Journal: Neuroscience

Article Title: A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability

doi: 10.1016/j.neuroscience.2018.03.030

Figure Lengend Snippet: c-Fos, pERK and pp38 are expressed in neuronal cells in Vc. Immunolabeling for c-Fos (A), pERK (B) and pp38 (C) co-localizes with neuronal marker NeuN in Vc. Representative figures are from brainstem slices incubated for 2 h in aCSF containing TTX plus XCL1 (see Methods) prior to processing for immunohistochemistry. (A–C) Confocal images of 50-μm transverse sections of brainstem caudal to obex. (A) Co-localization of c-Fos (red) and NeuN (green) in Vc. (B) Co-localization of pERK (red) and NeuN (green) in Vc. (C) Co-localization of pp38 (red) and NeuN (green) in Vc. In (A–C) the single staining for each antibody and the merged image are shown from left to right; co-localization appears in yellow. Scale bars = 10 μm.

Article Snippet: Slices were transferred to a holding chamber, where they were submerged in oxygenated aCSF maintained at 35 °C and incubated for 1 h. The slices were then incubated for 2 h with one of the following, diluted in aCSF: mouse recombinant XCL1 (0.1 µM; Sigma–Aldrich, UK); XCL1 antagonist vMIP-II (0.1 µM; R&D Systems, Minneapolis, MN, USA); normal aCSF; or vMIP-II (0.1 µM) plus XCL1 (0.1 µM) prior to the electrophysiological recordings.

Techniques: Immunolabeling, Marker, Incubation, Immunohistochemistry, Staining

Journal: Neuroscience

Article Title: A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability

doi: 10.1016/j.neuroscience.2018.03.030

Figure Lengend Snippet: XCL1 acting via XCR1 increases neuronal excitability in Vc. (A) Exemplar raw data trace from a single trigeminal brainstem slice showing: low-level spontaneous subthreshold voltage oscillations recorded in Vc in vitro in control aCSF (top trace); enhanced ongoing 4- to 12-Hz oscillatory activity in a single brainstem slice incubated in XCL1 (0.1 μM, 2 h) (upper middle trace); reduced intensity of XCL1-induced oscillatory activity by co-incubation of XCL1 and the antagonist vMIP-II (0.1 μM, 2 h) (lower middle trace). (B) Power spectra derived from raw data shown in (A) of low-amplitude rhythmic oscillations reveal a dominant frequency within 4- to 12-Hz frequency band and enhanced 4- to 12-Hz activity after exposure to XCL1 that is reduced by vMIP-II. Note that, relative to the drug-free control, MIP-II alone did not enhance baseline oscillatory activity. (C) The peak power amplitude and power area of the 4- to 12-Hz rhythmic activity was significantly increased within Vc in slices bathed in XCL1 ( p < 0.01); this effect was blocked by vMIP-II. Data are expressed as mean ± SEM and the number of slices used for each is shown in parenthesis on the x -axis (ANOVA with Dunnett’s post-hoc test).

Article Snippet: Slices were transferred to a holding chamber, where they were submerged in oxygenated aCSF maintained at 35 °C and incubated for 1 h. The slices were then incubated for 2 h with one of the following, diluted in aCSF: mouse recombinant XCL1 (0.1 µM; Sigma–Aldrich, UK); XCL1 antagonist vMIP-II (0.1 µM; R&D Systems, Minneapolis, MN, USA); normal aCSF; or vMIP-II (0.1 µM) plus XCL1 (0.1 µM) prior to the electrophysiological recordings.

Techniques: In Vitro, Activity Assay, Incubation, Derivative Assay