Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting Influenza Virus Hemagglutinin to Xcr1 + Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

doi: 10.4049/jimmunol.1601881

Figure Lengend Snippet: FIGURE 1. Human XCL1 and XCL2 vac- cibodies cross-react with murine Xcr1+ DCs. (A) Human XCR1+ DCs generated in vitro from cord blood CD34+ cells and identified by CD11clowBDCA3high expression were evaluated for binding of the human XCL vaccibodies. (B) Single-cell suspensions of BALB/c splenocytes were stained, and DCs defined as Lin2MHC-IIhighCD11chigh cells. DCs were separated into CD11b+ (cDC2) and CD24+ (cDC1) populations, and evaluated for binding of hXCL1-, hXCL2, or mXcl1- mCherry vaccibodies as compared with neg- ative control NIP-mCherry vaccibodies. (C) BM-derived Flt3L DCs from BALB/c mice were analyzed for binding to vaccibody pro- teins by flow cytometry. DCs were defined as CD45R/B2202CD11c+ cells, and further split into CD11b+ (cDC2) and CD24+ (cDC1) populations, and evaluated for binding to vaccibodies as in (B). (A) Data shown are representative of a single experiment with four separate DC preparations. Data shown are representative of two (B) or more than three (C) independent experiments.

Article Snippet: Abs against human XCL1 were obtained from R&D Systems (MAB6951) and mXcl1 from Lifespan BioSciences (Nottingham, U.K.) (C-16241).

Techniques: Generated, In Vitro, Expressing, Binding Assay, Staining, Control, Derivative Assay, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting Influenza Virus Hemagglutinin to Xcr1 + Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

doi: 10.4049/jimmunol.1601881

Figure Lengend Snippet: FIGURE 2. Human XCL1 and XCL2 vaccibodies provide equal short-term and better long-term protection against a lethal influenza challenge as compared with murine Xcl1 vaccibodies. (A) BALB/c mice were DNA immunized by intradermal injection followed by electroporation with plasmids coding for mXcl1-HA, hXCL1-HA, hXCL2-HA, NIP-HA vaccibodies, HA alone, or NaCl, and challenged with a 50xLD50 dose of PR8 virus 2 wk later. Weight loss was monitored as a measure of disease progression. (B) Survival plot of the mice in (A). (C) Mice were immunized as in (A) and challenged with a 5xLD50 after 26 wk. (A and C) Data shown are mean 6 SEM. (A–C) Data shown are representative of two independent experiments. (A and B) n = 6–12 (C) n = 9–10. (A and C) Two-way ANOVA, (B) Mantel–Cox, *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Abs against human XCL1 were obtained from R&D Systems (MAB6951) and mXcl1 from Lifespan BioSciences (Nottingham, U.K.) (C-16241).

Techniques: Injection, Electroporation, Virus, Biomarker Discovery

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting Influenza Virus Hemagglutinin to Xcr1 + Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

doi: 10.4049/jimmunol.1601881

Figure Lengend Snippet: FIGURE 3. Human XCL1 and XCL2 vaccibodies induce strong and long-lasting Ab responses after immunization. BALB/c mice were vaccinated as described in Fig. 2 and serum levels of HA-specific IgG determined longitudinally by ELISA (A). (B) HA-specific ASC in BM 6 wk after vaccination was measured by ELISPOT. (C and D) IFN-g– secreting splenocytes in response to stimulation with the HA-derived MHC- II restricted peptide HNTNGVTAAC- SHEG (C) or the MHC-I restricted peptide IYSTVASSL (D) 1 wk after vaccination. (E) HA-specific IgG1, IgG2a, and IgG2b responses in serum 15 wk after vaccination as measured by ELISA. (A–D) Data shown are mean 6 SEM. (A and E) Data shown are rep- resentative of two independent exper- iments, n = 9–10. (B–D) Pooled data from two independent experiments are shown, n = 8. (B–D) Unpaired t test (two-tailed), (E) Mann–Whitney (two- tailed), *p , 0.05, **p , 0.01.

Article Snippet: Abs against human XCL1 were obtained from R&D Systems (MAB6951) and mXcl1 from Lifespan BioSciences (Nottingham, U.K.) (C-16241).

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting Influenza Virus Hemagglutinin to Xcr1 + Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

doi: 10.4049/jimmunol.1601881

Figure Lengend Snippet: FIGURE 4. Vaccination with human XCL1 and XCL2 vaccibodies results in protective Ab responses. (A) BALB/c mice were vaccinated as in Fig. 2 and the avidity of HA-specific IgG responses after 5 wk was measured by ELISA. (B) HI assay performed on serum samples harvested 5 wk after immunization. (C) IC50 titers determined by microneutralization assay on serum samples harvested at 5, 10, 15, 20, and 25 wk after immunization. (D) Naive mice received 200 ml of pooled sera i.v. from mice vaccinated 6 wk earlier, and were challenged with a 5xLD50 dose of PR8 virus the following day. Disease progression was monitored by weight loss. (E) Survival plot of the mice in (D). (A–D) Data shown are mean 6 SEM. (A) Data shown are from a single experiment, (B) pooled from two independent experiments, (C) representative of two independent experiments or (D and E) pooled from two independent experiments. (A) n = 9–10 mice per group, (B) n = 7–9 mice per group, (C) sera is pooled from two mice, n = 5 for each group, except hXCL2-HA where n = 4, (D and E) n = 11 mice for each recipient group. (A and B) Unpaired t test (two-tailed), (C and D) two-way ANOVA and (E) Mantel–Cox, *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Abs against human XCL1 were obtained from R&D Systems (MAB6951) and mXcl1 from Lifespan BioSciences (Nottingham, U.K.) (C-16241).

Techniques: Enzyme-linked Immunosorbent Assay, HI Assay, Microneutralization Assay, Virus, Biomarker Discovery, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting Influenza Virus Hemagglutinin to Xcr1 + Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

doi: 10.4049/jimmunol.1601881

Figure Lengend Snippet: FIGURE 6. Human XCL1 and XCL2 vaccibodies are not endocytosed and remain on the surface of Xcr1+ DCs. (A) Flt3L DCs were incubated in transwell plates with NIP-, mXcl1-, hXCL1- or hXCL2-mCherry vaccibodies, and the migrated cells analyzed by flow cytometry. The migration index calculated as the ratio of CD24+ population (cDC1) relative to the CD11b+ population (cDC2) of the migrated cells. (B) Chemotaxis assay performed on human PBMC incubated with NIP- or hXCL1-mCherry. Migration index is defined as the percentage of CLEC9A+SIRPa2 DCs migrated after incubation with vaccibody protein divided by initial percentage of CLEC9A+SIRPa2 DCs in sample. (C) Internalization of mCherry vaccibodies incubated with Flt3L DCs at 37˚C for 0, 15, or 30 min. The cells were then incubated with a biotinylated anti-mCherry and streptavidin- allophycocyanin-Cy7 conjugate to stain for vaccibodies on the surface of the cells, and subsequently analyzed by flow cytometry. Internalization index is the ratio of mean fluorescence intensity for the allophycocyanin-Cy7 and mCherry signals. (D) Images of Xcr1+ Flt3L DCs incubated with vaccibody proteins for 0 and 30 min at 37˚C obtained by ImageStream. (E) Internalization of the Xcr1 receptor after incubation with mXcl1-, hXCL1- and hXCL2 vaccibodies for 0, 15, or 30 min at 37˚C as determined by ImageStream. The plotted values are relative to internalization after incubation with NIP-vaccibodies. (A–C and E) Data shown are mean 6 SEM. (A) Data shown are representative of two independent experiments with n = 3, (B) from one experiment with three separate donors, (C) pooled from two independent experiments with n = 5, (D) representative of three in- dependent experiments or (E) pooled from three independent experiments with n = 3. (C and E) Unpaired t test (two-tailed), *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Abs against human XCL1 were obtained from R&D Systems (MAB6951) and mXcl1 from Lifespan BioSciences (Nottingham, U.K.) (C-16241).

Techniques: Incubation, Cytometry, Migration, Chemotaxis Assay, Staining, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Superior antigen cross-presentation and XCR1 expression define human CD11c + CD141 + cells as homologues of mouse CD8 + dendritic cells

doi: 10.1084/jem.20100348

Figure Lengend Snippet: XCL1 induces a [Ca 2+ ] i signal in CD141 + DCs. CD141 + DCs were flow sorted to a purity >98.7%, immobilized on poly– l -lysine–coated glass coverslips, and loaded with 2 µM fura-2/AM. Cells were imaged in a monochromator-assisted digital video imaging system and challenged with 1 µg/ml XCL1 as indicated (left arrow). Subsequently, the same cells were challenged again with a mixture of 100 ng/ml CCL2, 200 ng/ml CCL21, 200 ng/ml CXCL9, and 1 ng/ml CX3CL1 used as a positive control (right arrow). The data shown represent [Ca 2+ ] i concentrations of 300 single cells (gray lines) measured in two independent experiments. The mean [Ca 2+ ] i signal averaged over all cells responding to XCL1 is indicated (black line).

Article Snippet: The lower chamber was filled with chemotaxis medium containing recombinant human XCL1 (R&D Systems) or any of the chemokines CCL2 (100 ng/ml), CCL21 (200 ng/ml), CX3CL1 (1 ng/ml), CXCL12 (200 ng/ml for T cells, B cells, NK cells, and monocytes; 100 ng/ml for pDCs), and CXCL8 (100 ng/ml; all from R&D Systems), and the cells were incubated for 150 min at 37°C in 5% CO 2 .

Techniques: Imaging, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Superior antigen cross-presentation and XCR1 expression define human CD11c + CD141 + cells as homologues of mouse CD8 + dendritic cells

doi: 10.1084/jem.20100348

Figure Lengend Snippet: XCL1 selectively induces chemotaxis in CD141 + DCs. (A) A mixture of highly purified, flow-sorted DC subtypes (20% CD141 + , 40% CD16 + , and 40% CD1c + DCs; Input DC) was tested for migration in response to medium alone or to serial dilutions of XCL1 (10–5,000 ng/ml) in a Transwell system. A combination of the chemokines CCL2, CCL21, and CX3CL1 was used as a positive control for the DC subsets (Migrated DC). The absolute numbers of CD141 + , CD1c + , and CD16 + DCs in input and migrated cell populations are truly represented in the dot plots, because all cells within a defined volume were included in the analysis in each instance. (B) Proportion of migrated CD1c + , CD16 + , and CD141 + DCs in the experiment shown in A. (C) Proportion of migrated pDCs, monocytes, granulocytes, T cells, B cells, and NK cells in response to XCL1 (10–1,000 ng/ml) or the chemokines CXCL12 and CXCL8, which were used as positive controls. For migration assays of B cells, NK cells, and monocytes, PBMCs were magnetically depleted of T cells, and for T cell migration, PBMCs were used directly. For migration assays of granulocytes, whole blood cells were used after erythrocyte lysis with ACK buffer, and pDCs were magnetically enriched from PBMCs with the Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec). All experiments with DCs were performed three times; all other populations were assayed twice. Error bars represent means ± SEM.

Article Snippet: The lower chamber was filled with chemotaxis medium containing recombinant human XCL1 (R&D Systems) or any of the chemokines CCL2 (100 ng/ml), CCL21 (200 ng/ml), CX3CL1 (1 ng/ml), CXCL12 (200 ng/ml for T cells, B cells, NK cells, and monocytes; 100 ng/ml for pDCs), and CXCL8 (100 ng/ml; all from R&D Systems), and the cells were incubated for 150 min at 37°C in 5% CO 2 .

Techniques: Chemotaxis Assay, Purification, Migration, Positive Control, Lysis, Cell Isolation

Journal: The Journal of Experimental Medicine

Article Title: Superior antigen cross-presentation and XCR1 expression define human CD11c + CD141 + cells as homologues of mouse CD8 + dendritic cells

doi: 10.1084/jem.20100348

Figure Lengend Snippet: Involvement of the XCL1–XCR1 communication axis in the innate and adaptive cytotoxic responses to cross-presented microbial and tumor antigens. Secretion of the chemokine XCL1 by activated NK cells specifically attracts XCR1-expressing DCs capable of antigen cross-presentation. This ensures an effective communication between these cells in the innate phase of the immune response. In the adaptive phase, secretion of XCL1 by activated CD8 + T cells optimizes the communication with antigen cross-presenting DCs and facilitates the differentiation of CD8 + T cells to cytotoxic cells.

Article Snippet: The lower chamber was filled with chemotaxis medium containing recombinant human XCL1 (R&D Systems) or any of the chemokines CCL2 (100 ng/ml), CCL21 (200 ng/ml), CX3CL1 (1 ng/ml), CXCL12 (200 ng/ml for T cells, B cells, NK cells, and monocytes; 100 ng/ml for pDCs), and CXCL8 (100 ng/ml; all from R&D Systems), and the cells were incubated for 150 min at 37°C in 5% CO 2 .

Techniques: Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: CXCL-8 secretion varied with glycan alterations. (A) Experimental workflow for assessing pro-inflammatory cytokine secretion of glycosylation mutants compared to wildtype H. pylori . Image was created using BioRender. (B) The glycoprotein mutant with elaborated LPS structures, Δ579, induced increased CXCL-8 secretion from gastric epithelial cells relative to wildtype. (C) The LPS mutant, Δwzk, induced decreased CXCL-8 secretion from gastric epithelial cells relative to wildtype. (D–I) Variable impact on CXCL-8 secretion was observed with glycoprotein mutants Δ580 and Δ1179, and LPS mutant, ΔwaaL, compared to wildtype depending on the coculture experiment. Error bars reflect technical replicates. Tukey’s multiple comparison test one-way ANOVA was used. (**P< 0.01, ***P< 0.001, ****P< 0.0001, ns, not significant). Data are representative of independent replicate experiments (n > 3) that exhibited the same findings.

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Glycoproteomics, Mutagenesis, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: Immature dendritic cell cytokine secretion changed when challenged by H. pylori glycosylation mutants. (A) Experimental workflow used to assess TNF-α, IL-6, IL-1β, and IL-10 secretion from iDCs following challenge for 24-hours with glycosylation mutants or wildtype bacteria. Image was created using BioRender. Datasets from two different iDC challenge experiments are shown, with (B–E) collected in one experiment and (F–I) collected in a second experiment. Δ580.1 and Δ 580.2, as well as Δ1179.1 and Δ1179.2, represent different freeze lots of the same strain. Error bars reflect technical replicates. Tukey’s multiple comparison test one-way ANOVA was used. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, ns, not significant).

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Glycoproteomics, Bacteria, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: The glycocalyx was probed to assess how Lewis Y expression and LPS elaboration correlate to cytokine secretion by immune cells. (A) Western blot analysis reveals robust Lewis Y expression in Δ579 and Δ580.2. The strain samples used in these experiments correspond to those used in iDC challenge experiments for the dataset shown in Figure 5 . (B, C) Analysis of LPS by gel electrophoresis reveals that Δwzk and ΔwaaL synthesize only truncated, low molecular weight LPS relative to wildtype H. pylori , whereas Δ579 appears to synthesize LPS with somewhat more prominent bands at higher molecular weights (e.g., >29kDa) than wildtype H. pylori . (B) The strain samples used in these experiments correspond to those used in dendritic cell challenge experiments for the dataset shown in Figure 5 . Δ580.1 and Δ 580.2 represent different freeze lots of the same strain. (C) Analysis of LPS by gel electrophoresis for strain samples used in dendritic cell challenge experiments for the dataset shown in Supplementary Figure S3 . Δ1179.1 and Δ1179.2 represent different freeze lots of the same strain and appear to have comparable LPS fingerprints.

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Expressing, Western Blot, Nucleic Acid Electrophoresis, Molecular Weight

Journal: PLoS Medicine

Article Title: Marked Differences in Human Melanoma Antigen-Specific T Cell Responsiveness after Vaccination Using a Functional Microarray

doi: 10.1371/journal.pmed.0020265

Figure Lengend Snippet: Eleven samples taken from patients enrolled in peptide vaccine trials were analyzed on pMHC functional microarrays. Patients received eight subcutaneous injections of peptides gp100 209–2M, MART1 M26, and tyrosinase 370D, along with adjuvant in a 6-mo period. Leukopheresis samples were collected after the eighth injection. Sample 10794 was collected from the same patient as 10735 after month 12. Functional profiles were generated by incubating patient CD8+ T lymphocytes on pMHC functional microarrays for 24 h at 37 °C and detecting the secreted factors with biotinylated secondary antibodies and streptavidin-phycoerythrin. Data were analyzed by automated fluorescence microscopy. Responses were scored on a five-point scale (0 to 4 bars), reflecting number of responders and overall fluorescent signal intensity per spot . Four bars indicate a strong response, and “0” indicates lack of a response. Each spot was printed in triplicate and analyzed individually. Patient clinical data are listed, including age and sex ( “ID”), stage of disease at enrollment (“Stage”), and outcome at follow-up (“Outcome”). Column labeled “IL12” specifies IL-12 adjuvant doses. Patient 10713 also received GM-CSF in addition to IL-12 adjuvant. Other secreted factors not shown include IL-4, IL-5, IL-10, IL-12p70, IL-1b, IL-3, IL-7, IL-13, IL-15, IL-17, lymphotactin, IP-10/CXCL10, TNFβ, VEGF, VEGF-D and granzyme A due to either lack of detectable secretion or limited analysis performed on only a fraction of the samples. In vitro restimulated cell lines directed against gp100 209 (132.2), MART1 M27 (461.30), or CMV pp65 495 (CMV94.3) were bound and secreted factors in response to gp100, MART1, and CMV (unpublished data), respectively.

Article Snippet: Antibodies against human CD8 (HIT8a), HLA-A2 (BB7.2), IFNγ (NIB42, 4S.B3), TNFα (MAb1, MAb11), granzyme B (2CF/F5, GB11), GM-CSF (BVD2-23B6, BVD2-21C11), IL-2 (5344.111, B33–2), IL-4 (8D4–8, MP4-25D2), IL-5 (TRFK5, JES1-5A10), IL-10 (JES3-9D7, JES3-12G8), and IL-12p70 (20C2, C8.6) were purchased from BD Pharmingen (San Diego, California, United States); antibodies against IL-1a (4414.141, pAb BAF200), IL-1b (8516.311. pAb BAF201), IL-3 (4815.211, pAb BAF203), IL-6 (6708, pAb BAF206), IL-7 (7417, pAb BAF207), IL-13 (32116, pAb BAF213), IL-15 (34593, pAb BAF247), IL-17 (41809, pAb BAF317), lymphotactin (109001, pAb BAF695), IP-10/CXCL10 (33036.211, pAb BAF266), TGF-β1 (9005, 27240), TNFβ (5807, pAb BAF211), vascular endothelial growth factor (VEGF; pAb AF293NA, pAb BAF293), and VEGF-D (78902, 78923) were purchased from R&D Systems (Minneapolis, Minnesota, United States), and granzyme A (CLB-GA29, CLB-GA28) was purchased from Research Diagnostics Incorporated (Flanders, New Jersey, United States).

Techniques: Functional Assay, Adjuvant, Injection, Generated, Fluorescence, Microscopy, Labeling, In Vitro

Journal: The Journal of rheumatology

Article Title: Expression and function of the C-class chemokine lymphotactin (XCL1) in Wegener's granulomatosis.

doi: 10.3899/jrheum.090244

Figure Lengend Snippet: Figure 2. Flow cytometric analysis results of XCL1 expression. PBMC isolated from WG patients and healthy controls were subjected to flow cytometric analysis after stimulation with PMA/ionomycin. XCL1 was expressed in a significantly greater proportion of CD4+ and CD8+ T cells in WG patients in comparison to controls (A). XCL1 was detected in WG patients in a significantly greater proportion of CD4+ T cells lacking the costimulatory molecule CD28 (p = 0.007) (B). In CD8+ T cells, XCL1 expression was found mainly within the CD8+CD28– T cell subpopulation in WG patients, but there was no statistically significant difference of frequencies of XCL1-expressing CD8+CD28– T cells between WG patients and controls (C). Comparing T cells from patients with active and inactive WG, a significant difference was detected between these patient groups in frequencies of XCL1-positive CD4+ T cells and CD8+ T cells (p < 0.001) (D).

Article Snippet: Cells were subsequently stimulated with human recombinant XCL1 (R&D Systems, Wiesbaden, Germany) at a concentration of 100 ng/ml for 24 h. To control the specificity of XCL1 action, anti-human XCL1 antibody (R&D Systems) was added to the control wells.

Techniques: Expressing, Isolation, Comparison

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