Journal: Cell metabolism

Article Title: Miro1 Marks Parkinson’s Disease Subset and Miro1 Reducer Rescues Neuron Loss in Parkinson’s Models.

doi: 10.1016/j.cmet.2019.08.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For fluorescent Western, blots were probed with Cy5-conjugated goat anti-mouse IgG (PA45009, GE Healthcare) at 1:5,000, and scanned at 635nm with a Molecular Dynamics Storm 860 Imager (Amersham BioSciences, Piscataway, NJ) in a linear range for fluorescent detection.

Techniques: Virus, Recombinant, Activity Assay, Software

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Effector machinery of CD57 +/ −CD45RA +/ − or CD57 +/ − CXCR5 +/ − CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Identification of CD57 + CXCR5 + T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57 + CD4 T cells in GCs. (C) Localization of CD57 + CXCR5 + CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, In Situ, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Chemotactic responses (A) and chemokine receptor expression (B) by CD57 +/2 CXCR5 +/ − CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, Chemotaxis Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Spontaneous B cell helper activity of CD57 +/ − CXCR5 +/ − CD4 T cells. Sorted CD57 + CXCR5 + , CD57 − CXCR5 + , and CXCR5 − CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Phenotype and effector function of circulating CXCR5 + T cells. (A) Surface phenotype of circulating naive or CXCR5 +/ − memory T cells. **Significant differences between CXCR5 + and CXCR5 − memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5 +/ − T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5 + T cells after T cell receptor activation. Naive (IgD + ) or memory (IgD − ) B cells (2 × 10 4 ) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10 3 , 5 × 10 3 , 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 10 5 ) of autologous T cells (CXCR5 − CD45RA + , CXCR5 + CD45RA − , or CXCR5 − CD45RA − ). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activation Assay, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 1 Cre-mediated recombination selectively depletes TLR4 on endothelial cells. Representative retinal whole mounts of TekCretdTomato mice stained with Iba-1 (green) in naïve mice (A–C) and LPS-challenged mice (E–G). tdTomato (magenta) was selectively expressed in retinal blood vessels and no co-localization with Iba-1 was detected. The yellow squares in C and G are magnified in D and H, respectively. Scale bars: 100 μm. Representative dot plots of single, live, TIE2posTLR4pos cells in the retinas of naïve TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice (I, J). The majority of TIE2pos cells (72.7%) in the TekCre−negTlr4loxP/loxP retinas express TLR4, while in the retinas of TekCre−posTlr4loxP/loxP mice only 6.4% of TIE2pos cells are TLR4pos (K; n = 3 mice per group). The data were analyzed with 2-tailed unpaired t-test, ***p < 0.001)

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Staining

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 2 Systemic LPS fails to trigger microglia activation and retinal vasodilation in endothelial Tlr4-deficient mice. Representative images of Iba-1-stained retinal whole mounts in naïve (A, G, M) and LPS-challenged (D, J, P) C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Iba-1pos cells located in the GCL-INL and the OPL are shown in magenta and white, respectively. Yellow arrows in D indicate the clustering of Iba-1pos cells around a retinal vessel. In naïve C57BL/6J mice, microglia cells have a ramified morphology throughout the retina (B, C). After the LPS challenge, Iba-1pos cells adopt a bushy or amoebic morphology and accumulate around retinal blood vessels in the GCL-INL (E). In the OPL, these cells have an amoebic or rod-like shape (F). The morphology of Iba-1pos cells before or after the LPS challenge in TekCre−negTlr4loxP/loxP mice was comparable to C57BL/6J mice (H, I, K, L). Individual Iba-1pos cells of TekCre−posTlr4loxP/loxP mice had a ramified morphology both in the naïve and the LPS-challenged group (N, O, Q, R). Quantification of average branch length (S; n = 4–5 mice per group), number of endpoints per cell (T; n = 4–5 mice per group) and Iba-1 occupied volume (U; n = 4–5 mice per group) on images obtained from retinal whole mounts. Reduced branch length, less endpoints per cell and increased Iba-1 occupied volume were detected in the C57BL/6J but not the endothelial Tlr4 knockout mice. Representative images of fluorescein angiographs at baseline (V, X) and after the LPS challenge (W, Y) in C57BL/6J and TekCre−posTlr4loxP/loxP mice, respectively and quantification of vein dilation (Z; n = 5–7 mice per group). Vein diameter was significantly increased in LPS-challenged C57BL/6J mice compared to baseline, but not in TekCre−posTlr4loxP/loxP mice. The quantification data are reported as mean ± SD. The data were analyzed separately for each genotype with 2-tailed unpaired t-test (S–U) or 2-tailed paired t-test (Z). **p < 0.01, ***p < 0.001. a Artery; v vein; GCL-INL ganglion cell layer-inner nuclear layer; OPL outer plexiform layer. Scale bars: left panel: 100 μm; middle and right panels: 50 μm

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Activation Assay, Staining, Knock-Out

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 3 Effect of LPS on microglia/macrophage numbers in the presence or absence of endothelial Tlr4. Representative dot plots of CD11bpos populations (A, B left panels) gated as CD11bposCD45low/neg microglia and CD11bposCD45hi monocyte-derived macrophages (A, B right panels) in naïve and LPS-challenged C57BL/6J and TekCre−posTlr4loxP/loxP mice, respectively. Quantification of CD45low/neg microglia and CD45hi macrophages expressed as a percentage of the total number of live cells in the samples (C, D) revealed an increase of both populations after the LPS challenge in C57BL/6J mice but not in TekCre−posTlr4loxP/loxP mice (n = 5 mice per genotype and experimental group). The data were analyzed separately for each genotype with 2-tailed unpaired t-test (***p < 0.001). Representative images of retinal sections stained with Iba-1 and ICAM-1 in naïve (E, G, I) and LPS-challenged (F, H, J) C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. ICAM-1 immunoreactivity was detected in LPS-challenged C57BL/6J and TekCre−negTlr4loxP/loxP mice but not in TekCre−posTlr4loxP/loxP mice. Scale bars: 200 μm. GCL Ganglion cells layer; INL inner nuclear layer; IPL inner plexiform layer; ONL outer nuclear layer; OPL outer plexiform layer

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Derivative Assay, Staining

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 4 Endothelial Tlr4 depletion rescue the retina from LPS-induced visual dysfunction. Representative waveforms at baseline (A, C, E) and after the LPS challenge (B, D, F) under scotopic conditions in C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Comparison of a- (G, I, K) and b-wave amplitudes (H, J, L) between baseline and LPS in C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Reduced mean a- and b-wave amplitudes were recorded in C57BL/6J and TekCre−negTlr4loxP/loxP mice after the LPS challenge. In TekCre−posTlr4loxP/loxP mice, mean a- and b-wave amplitudes after the LPS challenge were comparable to baseline levels. The data were analyzed with repeated measures 2-way ANOVA followed by Sidak’s post hoc analysis (n = 4–5 mice per genotype, one eye per mouse; *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Comparison

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 6 Proposed mechanism of systemic LPS-induced retinal microglia activation and retinal dysfunction. A. The inner BRB (iBRB) is composed by a deep, an intermediate, and a superficial vascular plexus. In the healthy retina, endothelial cells, that line the blood vessels, are connected by tight junctions forming an effective vascular barrier. Endothelial cells bear TLR4 receptors and they are surrounded by pericytes and perivascular macrophages. Microglia resides in close proximity to the vasculature, while monocytes circulate in the bloodstream. B. Upon the systemic LPS challenge, LPS binds to TLR4 located on circulating monocytes and endothelial cells (1), and induces the expression of the adhesion molecule ICAM-1 from the latter (2). Activated endothelial cells release cytokines and chemokines (3), which may act as chemoattractants causing microglia migration towards the affected vasculature (4). Subsequently, monocyte-derived macrophages are entering the retina through the disrupted BRB and AIF-1 – ICAM-1 interactions (5). Migration of microglia away from retinal neurons may account for disruption of ribbon synapses and impaired retinal function (6). EC endothelial cell; GCL ganglion cell layer; iBRB inner blood retinal barrier; ICAM-1 intercellular molecule 1; INL inner nuclear layer; IPL inner plexiform layer; LFA-1 lymphocyte function-associated antigen 1; LPS lipopolysaccharide; M microglia; MDMs monocyte-derived macrophages; ONL outer nuclear layer; OPL outer plexiform layer; PRs photoreceptors; PVMs perivascular macrophages; RSs ribbon synapses; TJs tight junctions; TLR4 Toll-like receptor 4

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Activation Assay, Expressing, Migration, Derivative Assay, Disruption