Journal: Stem Cell Research & Therapy

Article Title: CCL11 promotes migration and proliferation of mouse neural progenitor cells

doi: 10.1186/s13287-017-0474-9

Figure Lengend Snippet: Isolation of NPCs and migration assay. a The neurosphere from embryonic and infant mouse brains. Genes involved in proliferation and multipotency were expressed in cultured NPCs. Scale bar = 50 μm. b Flow-cytometric analysis for NPCs. The left panel showed an isotype control mouse IgM antibody. The right panel showed that PSA-NCAM was expressed in NPCs. c Immunostaining of Dcx for embryonic NPCs. Dcx-positive cells showed spindle cell morphology. Green : Dcx; blue : DAPI. Scale bar = 30 μm. d Migration assay using μ-Slide Chemotaxis. NPCs were seeded at 3 × 10 6 cells/ml in the shaded area. Chemokine was injected through the indicated port. Time-lapse imaging and tracking of NPCs with CCL11 ( blue line ). Scale bar = 100 μm. e , f The trajectory of randomly selected embryonic ( e ) and infant ( f ) NPCs in migration assay. Each color represents the trajectory of an individual cell. The y-axis and the x-axis represents migration distance (μm). The minus (leftward) direction of cell movement is defined as chemokine-induced migration. g The distance and end point of embryonic NPCs migration. CCL11 significantly increased the migration distance of NPCs compared with the control (PBS) and other chemokines. The data are presented as the mean of end points ± S.D. The migration assay was repeated three independent times per chemokine. * P < 0.05, ** P < 0.01. h The end point of trajectory of the embryonic NPCs ( E-NPC ) and infant NPCs ( I-NPC ) derived from the injury and intact side of mouse brain using CCL11. * P < 0.05. n.s. not significant

Article Snippet: A neutralizing rat monoclonal CCL11 antibody (R&D Systems) at a dose of 25 μg/mouse was administrated twice at 6 and 24 hours after brain injury.

Techniques: Isolation, Migration, Cell Culture, Control, Immunostaining, Chemotaxis Assay, Injection, Imaging, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: CCL11 promotes migration and proliferation of mouse neural progenitor cells

doi: 10.1186/s13287-017-0474-9

Figure Lengend Snippet: The cell culture assay for NPCs. a Proliferation of embryonic NPCs was enhanced after incubation with 2 μg/ml CCL11 at 3 days post-treatment. Other CC chemokines neither formed the neurospheres nor enhanced NSC proliferation. The data are presented as the mean absorbance of three replicates ± S.D. ** P < 0.01. b The concentration-dependent effect of CCL11 on NPC proliferation. High concentration of CCL11 (2 μg/ml, 5 μg/ml) promoted NPC proliferation. ** P < 0.01 vs control (PBS). c NPCs treated with 2 μg/ml CCL11 formed neurospheres at 3 days post-treatment. Scale bar = 50 μm. d CCL11 (2 μg/ml) enhanced proliferation of infant NPCs derived from both injury and intact sides of the mouse brain. The data are presented as the mean absorbance of three replicates ± S.D. * P < 0.05. e , f The CCR3 receptor antagonist SB 297006 blocked the CCL11-induced NPC proliferation and neurosphere formation. Scale bar = 50 μm. * P < 0.05, ** P < 0.01. g Merged image of double immunostaining of Dcx and CCR3 for embryonic NPCs. Green : Dcx; red : CCR3, blue : DAPI. Scale bar = 30 μm. h RT-PCR analysis of CCR3 expression in NPCs. CCR3 are expressed in embryonic and infant NPCs

Article Snippet: A neutralizing rat monoclonal CCL11 antibody (R&D Systems) at a dose of 25 μg/mouse was administrated twice at 6 and 24 hours after brain injury.

Techniques: Cell Culture, Incubation, Concentration Assay, Control, Derivative Assay, Double Immunostaining, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: The Journal of Experimental Medicine

Article Title: ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

doi: 10.1084/jem.20201142

Figure Lengend Snippet: Increased VAT-T R accumulation in the absence of ICOS signaling is associated with elevated CCR3 expression. (A) ST2 expression in splenic T R s ( n = 3–5 per group from eight independent experiments). (B) ST2 expression in CD45.1 + and CD45.2 + donor splenic T R s in WT:YF and WT:KO chimeric mice. Lines connect CD45.1 + and CD45.2 + donor splenic T R s within the same chimera ( n = 3–5 chimeric mice per group from three independent experiments). (C) Expression of CCR3 in VAT-T R s in mice ≤8 wk and >8 wk of age ( n = 3–5 per group from seven independent experiments). (D) Expression of CCR2 and CCR3 by VAT-T R s ( n = 3–5 per group from two independent experiments). DP, CCR2 + CCR3 + ; DN, CCR2 − CCR3 − . (E) Expression of indicated CCR3 ligands in total VAT normalized to Tbp as measured by qPCR (n.d. indicates not detected; n = 5 per group). (F) Schematic of in vitro culture experiments examining the impact of ICOS signaling on CCR3 expression (left). Graphs indicating fold change in T R frequency of CD4 + cells and %CCR3 + of T R s between individual culture samples stimulated (stim) with or without αICOS for 2 d (middle). Representative flow cytometry plots with frequency of CCR3 + T R s after 2 d in specified culture conditions (right; n = 1–3 per group from three independent experiments). (G) Left: Representative flow cytometry plots indicating VAT-T R frequency with or without CCL11/24 blockade. Graphs summarize T R frequencies in indicated tissues after 2 wk ( n = 3–4 per group). Mice were age matched within independent experiments and collectively; pooled data are from experiments using male mice aged 8–16 wk unless otherwise indicated. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A and C); two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (B); and two-tailed Student’s t test (F and G). All data are presented as mean values ± SD.

Article Snippet: Mice aged 8–10 wk were given 0.75 µg/g body weight αCCL11 and αCCL24 (MAB420 and MAB528; R&D Systems) or an equivalent amount of rat IgG (Sigma) diluted in PBS by i.p. injection on days 0, 5, and 10 and sacrificed for analysis on day 13.

Techniques: Expressing, In Vitro, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

doi: 10.1084/jem.20201142

Figure Lengend Snippet: Increased accumulation of CCR3 + T R s in the absence of ICOS signaling is specific to VAT (goes with ). (A) CCR2 expression by splenic (top) and VAT-T R s (bottom) as measured by flow cytometry ( n = 3–5 per group from two independent experiments). (B) Expression of CCR3 in splenic T R s in mice ≤8 wk and >8 wk of age ( n = 3–5 per group from seven independent experiments). (C) Expression of CCR3 by gated CD45.1 + and CD45.2 + donor VAT-T R s in chimeric mice. Graphs summarize CCR3 expression by donor T R s in VAT and spleen. Line connects point representing CD45.1 + and CD45.2 + cells within the same chimeric mouse ( n = 2–4 per group from two independent experiments). (D) CCR3 expression by tissue-localized skin T R s as measured by flow cytometry ( n = 2–4 per group from two independent experiments). (E) CCR3 expression by CD45.1 + and CD45.2 + donor T R s within the same chimeric mouse in indicated tissues. Line connects CD45.1 + and CD45.2 + cells within the same chimeric mouse ( n = 2–4 per group from two independent experiments). (F) Frequency of tissue-restricted VAT eosinophils with and without in vivo CCL11/24 blockade as measured by flow cytometry ( n = 3 or 4 mice per group). Mice were age matched within individual experiments, and pooled data are from experiments using 8–16-wk-old male mice. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A, B, and D); two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (C and E); and two-tailed Student’s t test (F). All data are presented as mean values ± SD. LPL, lamina propria lymphocyte.

Article Snippet: Mice aged 8–10 wk were given 0.75 µg/g body weight αCCL11 and αCCL24 (MAB420 and MAB528; R&D Systems) or an equivalent amount of rat IgG (Sigma) diluted in PBS by i.p. injection on days 0, 5, and 10 and sacrificed for analysis on day 13.

Techniques: Expressing, Flow Cytometry, In Vivo, Two Tailed Test