anti mouse cd11b pe cy5  (Bioss)

Journal: The Journal of Clinical Investigation

Article Title: Neutrophil-microglia interaction drives motor dysfunction in a neuromyelitis optica model induced by subarachnoid AQP4-IgG

doi: 10.1172/JCI199706

Figure Lengend Snippet: ( A ) Timeline for injecting neutrophil-depleting anti-Ly6G-IgG or isotype control-IgG (100 mg/kg, i.p.), inserting lumbar subarachnoid catheter, infusing AQP4-IgG, and rotarod testing. ( B ) Flow cytometry confirms neutrophil ablation efficiency (percentage CD45 + CD11b + Gr1 + MPO + cells among peripheral CD45 + CD11b + cells). ( C ) (Upper) t-SNE analysis of CD45 + immune cell subtypes from lumbar spinal cords of control and neutrophil-depleted mice. (Lower) Quantification of data in B (3 mice/group). ( D ) Representative confocal images of neutrophils in lungs of mice receiving neutrophil-depleting anti-Ly6G-IgG or isotype control-IgG (3 mice/group). ( E ) Microglial activation, reflected by Cx3cr1GFP signal, in corresponding lumbar cord regions of mice without and with neutrophil ablation (by Ly6G-IgG or isotype control-IgG) after 3 days’ infusion with normal control mouse IgG or AQP4-IgG. ( F ) Quantification of microglia-occupied areas in E ( n = 4–5 mice per group). ( G ) Motor function, reflected by rotarod test, in neutropenic mice (anti-Ly6G-IgG–treated) and non-neutrophil-ablated (isotype control-IgG–treated) during 5 days’ infusion of AQP4-IgG or normal control mouse IgG (0.1 μg/μL; time: F (2.415, 28.98) = 4.838, P = 0.0113; treatment: F (2, 12) = 12.46, P = 0.0012; interaction: F (10, 60) = 7.100, P < 0.0001; n = 5 mice per group). ( H ) Experimental design: WT mice were continuously infused with Ctrl-IgG or AQP4-IgG by osmotic pumps for 7 days from day 0; infusion was discontinued at day 8. ( I ) Motor impairment worsened progressively in AQP4-IgG recipients, with nadir at day 8. Continued rotarod testing for another 3 weeks showed progressive motor recovery from day 8. ( J ) Correlations between microglial activation state (lumbar microglial area) and latency to fall in rotarod test. Simple linear regression (1 dot represents 1 mouse at day 3 of IgG infusion). Statistics: C used t test; Tukey’s post hoc multiple comparisons test (1-way ANOVA) in F ; 2-way repeated measures ANOVA with Holm-Šídák post hoc test in G and I .

Article Snippet: The following antibodies identified mature neutrophils (Gr1 hi CD11b + , Gr hi MPO + ) , immature myeloid cells (Gr1 int CD11b + ) , and immature neutrophils or granulocytic precursors (Gr1 int MPO + ) ( , ) in peripheral blood using spectral flow cytometry (Cytek Aurora, Cytek Biosciences): CD45-violetFluor 450 (Tonbo, 75-0451-U100, clone 30-F11, 1:1,000), CD45-PE-CF594 (BD Biosciences, 562420, clone 30-F11, 1:1,000), CD11b-PE-Cy5 (Tonbo, 55-0112-U100, clone M1/70, 1:1,000), Ly6G-APC-Cy7 (BioLegend, 127623, clone 1A8, 1:200), Ly6C-PerCP (BioLegend, 128028, clone HK1.4, 1:100), and Gr1-PE (BioLegend, 108408, clone RB6-8C5, 1:200).

Techniques: Control, Flow Cytometry, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Neutrophil-microglia interaction drives motor dysfunction in a neuromyelitis optica model induced by subarachnoid AQP4-IgG

doi: 10.1172/JCI199706

Figure Lengend Snippet: ( A and B ) Gray matter of AQP4-IgG recipient mice, C5a immunoreactivity was not seen in GFAP + astrocytes ( A ) and rarely seen in IBA1 + microglia/macrophages ( B ). ( C ) Triple immunostaining of lumbar cord of AQP4-IgG recipient mice revealed C5a in neutrophils (MPO + , left) and monocytes (CCR2-GFP + , enhanced by anti-GFP-IgG, right). ( D ) The percentage of spinal cord cells expressing C5a in AQP4-IgG-infused mice ( n = 4 mice per group). ( E ) Colocalization analysis of C5a and MPO signals in C using Zen software. ( F and G ) Confocal images of neutrophil (Ly6G, red) containing C5a (green in F ), and C5 (green in G ) after TNF-α incubation in vitro. Granule protein, MPO + , magenta; nuclear segmentation, DAPI + , blue. BF, bright-field. ( H ) Lumbar cords from IgG-infused mice were dissociated enzymatically and subjected to high parametric flow cytometric analysis. ( I ) t-SNE maps identifying CD11b + , Ly6G + , Cx3cr1 + , and C5aR1 + cells and their expression levels among CD45 + immune cells. ( J ) Public database ( Brain RNA-Seq ) documents that C5ar1 mRNA in normal mouse brain is predominantly expressed in microglia/macrophages. ( K ) Flow cytometric plot shows that neutrophils infiltrate the lumbar cord in WT mice and in C5ar1- deficient mice at day 3. ( L ) Quantification of the percentage of neutrophils (CD45 + CD11b + Ly6G + Ly6C – cells among CD45 + CD11b + cells) in K ( n = 4 mice per group). ( M ) Motor function, assessed by rotarod performance (latency to fall), in WT and C5ar1 –/– mice infused with AQP4-IgG or (only C5ar1 –/– mice) normal mouse IgG (0.1 μg/μL, n = 6 mice per group). ( N ) Motor function of mice assessed as rotarod fall latency; treatment: F (1, 6) = 9.961, P = 0.0197; n = 6–7 mice per group. Statistics: D used Tukey’s post hoc multiple comparisons test in (1-way ANOVA); t test in J ; 2-way repeated measures ANOVA with Holm-Šídák post hoc test in M and N .

Article Snippet: The following antibodies identified mature neutrophils (Gr1 hi CD11b + , Gr hi MPO + ) , immature myeloid cells (Gr1 int CD11b + ) , and immature neutrophils or granulocytic precursors (Gr1 int MPO + ) ( , ) in peripheral blood using spectral flow cytometry (Cytek Aurora, Cytek Biosciences): CD45-violetFluor 450 (Tonbo, 75-0451-U100, clone 30-F11, 1:1,000), CD45-PE-CF594 (BD Biosciences, 562420, clone 30-F11, 1:1,000), CD11b-PE-Cy5 (Tonbo, 55-0112-U100, clone M1/70, 1:1,000), Ly6G-APC-Cy7 (BioLegend, 127623, clone 1A8, 1:200), Ly6C-PerCP (BioLegend, 128028, clone HK1.4, 1:100), and Gr1-PE (BioLegend, 108408, clone RB6-8C5, 1:200).

Techniques: Triple Immunostaining, Expressing, Software, Incubation, In Vitro, RNA Sequencing

Journal: iScience

Article Title: Heightened innate immune state induced by viral vector leads to enhanced response to challenge and prolongs malaria vaccine protection

doi: 10.1016/j.isci.2024.111468

Figure Lengend Snippet: MCMV prime induces and maintains myeloid cell numbers BALB/c mice were infected with MCMV-BAC or MCMV-B5, as shown in (A) experimental schematic. Splenocytes were phenotyped at days 15 and 55 post-MCMV prime. Plots and graphs show percent and number of (B) dendritic cells and subsets (CD3 − MHCII hi CD11c hi and CD8α +/− ), (C) inflammatory monocytes (CD3 − MHCII + CD11b + Ly6C + ). (D) Plots and graphs show gating and quantification of IL-12-secreting CD8α +/− DCs. Data are representative of 3–7 animals per group. Mean shown with error bars representing SEM and analyzed using one-way ANOVA followed by Dunn’s multiple comparisons test. ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S3 .

Article Snippet: anti CD11b (M1/70) anti-mouse PE-Cy5 , ebioscience , Cat#15011283; RRID: AB_468714.

Techniques: Infection

Journal: iScience

Article Title: Heightened innate immune state induced by viral vector leads to enhanced response to challenge and prolongs malaria vaccine protection

doi: 10.1016/j.isci.2024.111468

Figure Lengend Snippet: IFN-γ induced by MCMV does not affect T cell phenotype but does increase DC number and IL-12p40 CTV-labeled B5 TCR Tg T cells were adoptively transferred into Thy1.1 (BALB/c) mice that were then infected with MCMV-B5 and received anti-IFN-γ antibody or isotype days 44–48 p.i., as shown in (A) experimental schematic. Splenocytes were phenotyped for (B–D) B5 TCR Tg (Thy1.2 + ) CD4 + T cells and (E–G) innate cells (dendritic cells, and inflammatory monocytes) and quantification of serum IL-12p40 level at day 50 p.i. (B) Plot, histogram, and graph show gating and quantification of CD4 + Thy1.2 + CTV − B5 T cells. (C) Plots and bar graph show MCMV-B5-specific effector and memory T cell numbers. (D) Plot and bar graph show the number within each MCMV-B5-specific Tmem (CD127 hi CD44 hi ) subset (Tcm, TemE, and TemL), as defined by CD27 and CD62L. (E) Plot and histogram show CD8α +/− DC population gating and quantification. (F) Graph shows serum IL-12p40 levels. (G) Plots and graphs show gating and quantification of inflammatory monocytes CD3 − MHCII + CD11b + Ly6C + . (H) Plots and graph show gating and quantification of CD3 − DX5 + MHCII − NK cells. Data shown are representative 5 mice per group and mean shown with error bars representing SEM. Student’s t test was used; ns, not significant ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S3 .

Article Snippet: anti CD11b (M1/70) anti-mouse PE-Cy5 , ebioscience , Cat#15011283; RRID: AB_468714.

Techniques: Labeling, Infection

Journal: iScience

Article Title: Heightened innate immune state induced by viral vector leads to enhanced response to challenge and prolongs malaria vaccine protection

doi: 10.1016/j.isci.2024.111468

Figure Lengend Snippet: IFN-γ induced by MCMV boosts and drives increase in CD8α + dendritic cells for long-term protection by live malaria vaccine Infection and drug-cure live vaccine combined with MCMV boosting strategy for protection to heterologous rechallenge testing effect of neutralizing IFN-γ on innate cells and challenge. BALB/c mice were given infection and drug-cure vaccine combined with MCMV boosting strategy for protection to heterologous rechallenge. Age-matched mice were infected with P. chabaudi AS and then treated with two non-curative doses of chloroquine (CQ). Then they received MCMV booster at day 140, and some received anti-IFN-γ antibody or isotype at indicated days (194, 196, 198) after P. chabaudi vaccination (p.v.). All mice were reinfected with P. chabaudi AJ strain at 200 days p.v., as shown in (A) experimental schematic. Graphs showing (B) parasitemia curves and (C) weight loss after P. chabaudi AJ challenge or infection. (B and C) ## represents significance between groups by ANOVA. Comparisons of parasitemia and weight loss in vaccinated (vaccine_MCMV_isotype and vaccine_MCMV_αIFN-γ) and non-vaccinated groups (No vaccine_MCMV and No vaccine_No MCMV) show a significant difference using ANOVA and Tukey’s multiple comparisons ( p < 0.01, for all time points of parasitemia (B) and day 9 p.c. for weight loss (C)). There is also a significant difference ( p < 0.05) in parasitemia (B) between vaccine_MCMV_isotype and vaccine_MCMV_αIFN-γ at day 5 post-challenge. (D) Graph of serum level of IgG specific to P. chabaudi AJ at indicated days (5, 7, and 15, relative to challenge). (E) Graph of proportion of polyclonal effector T cells (CD127 - ) after challenge (d15 p.c.) out of CD4 + . (F–J) As above, mice were vaccinated and boosted with MCMV at day 140 followed by anti-IFN-γ neutralization at indicated days (194, 196, 198), but not challenged, before spleen cell recovery for phenotyping at day 200 p.v., as shown in (F) experimental schematic. Plots and graphs show (G) gating and number of total DC population (CD3 − CD11c hi MHCII hi ) and (H) CD8α − or CD8α + DCs. (I) Graph of serum IL-12p40 level at day 200 p.v. (J) Plots and graphs show gating and quantification of inflammatory monocytes (iMo, CD3 − CD11b + Ly6C + ) and NK cells (CD3 − DX5 + MHCII − ). Data shown are representative of 5 animals per group. Mean shown with error bars representing SEM. Groups were analyzed using ANOVA followed by Tukey’s post-test or with Student’s t test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. See also Figure S6 .

Article Snippet: anti CD11b (M1/70) anti-mouse PE-Cy5 , ebioscience , Cat#15011283; RRID: AB_468714.

Techniques: Infection, Neutralization, Cell Recovery

Journal: iScience

Article Title: Heightened innate immune state induced by viral vector leads to enhanced response to challenge and prolongs malaria vaccine protection

doi: 10.1016/j.isci.2024.111468

Figure Lengend Snippet:

Article Snippet: anti CD11b (M1/70) anti-mouse PE-Cy5 , ebioscience , Cat#15011283; RRID: AB_468714.

Techniques: Control, Virus, Recombinant, Giemsa Stain, Cell Stimulation, Transfection, Software