bsa rat anti ly6g  (Bio X Cell)

Journal: bioRxiv

Article Title: Aspirin hastens resolution of skeletal muscle inflammation and promotes recovery of muscle strength following acute injury

doi: 10.64898/2026.04.21.719989

Figure Lengend Snippet: A: Representative histological (H & E) and immunofluorescence images of TA muscle cross-sections at day 1 post-injury following treatment with vehicle (VEH), indomethacin (INDO), aspirin (ASA), or a combination of INDO + ASA. Sections are stained for Ly6G (neutrophils, red), CD68 (total macrophages, green), CD206 (M2-like macrophages, red), with DAPI (nuclei, blue) and Laminin (LAM, white) to visualize fiber boundaries. B-E : Quantification of day 1 post-injury inflammatory markers including Ly6G + cell density ( B ), CD68 + cell density ( C ), the ratio of Ly6G + to total CD68 + cells ( D ), and the M1/M2 macrophage ratio ( E ). F: Representative H & E and immunofluorescence images at day 3 post-injury for the same treatment groups and markers. G-J: Quantitative analysis of inflammatory cell dynamics at day 3 post-injury, including Ly6G + cell density ( G ), CD68 + cell area as a percentage of total tissue ( H ), CD206 + cell densities ( I ), and the M1/M2 ratio ( J ). All data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Holm-Šídák post hoc tests. Groups labeled with different letters are significantly different from one another, while groups sharing a common letter are not significantly different.

Article Snippet: Primary antibodies used include MyHC type I [Developmental Studies Hybridoma Bank (DSHB), BA-D5c, 1:100], MyHC type IIA (DSHB, SC-71c, 1:100), MyHC type IIB (DSHB, BF-F3c, 1:100), eMHC (DSHB, F1.652s, 1:20), Ly6G (GR1) (Bio-Rad, MCA2387, 1:50), CD68 (Bio-Rad, MCA1957, 1:200), CD206 (Bio-Rad, MCA2387, 1:50), and laminin (Abcam, ab7463, 1:200).

Techniques: Immunofluorescence, Staining, Labeling

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: For immunofluorescence staining, sections were permeabilized and blocked (30 min), using 0.25% Triton X-100 in PBS containing 2.5% bovine serum albumin and 5% donkey serum, then exposed 16–24 hours at 4°C to the following primary antibodies in PBS containing 0.1% Triton X-100 (Sigma) and 1% BSA: rat anti-Ly6G (Bio X Cell; BE0075-1, clone 1A8, 1:500), human/mouse polyclonal goat anti-MPO (R&D Systems; AF3667; 1:800), rat anti-NE (R&D Systems, MAB4517-SP, clone 887105, 1:500), mouse anti-C5 (Hycult Biotech; HM1073, clone BB5.1, 1:500), rat anti-C5a (Invitrogen; MA5-23910, clone 295108, 1:300), goat polyclonal anti-GFP (Abcam; Ab6673, 1:1,000), polyclonal goat anti-CD31 (R&D Systems; AF3628, 1:500), rabbit antiIBA1 (Abcam; Ab178846 , clone EPR16588 , 1:500), polyclonal goat antiIBA1 (Wako; 011-27991, 1:500), guinea pig antiIBA1 (Synaptic Systems; 234 308, clone Gp311H9, 1:1,000), rabbit polyclonal anti-ChAT (Sigma-Aldrich, AB143; 1:100), rabbit polyclonal anti-P2Y12 (AnaSpec; AS-55043A; 1:1,000), human anti-HuD ( ) (from patients with paraneoplastic neurological autoimmunity related to small-cell lung carcinoma; 1:10,000), polyclonal goat anti-human/mouse/rat Galectin-3 antibody (R&D Systems; AF1197, NP_034835 , 1: 1,000), and mouse anti-4-HNE (R&D Systems; MAB3249-SP, clone 198960, 1:200).

Techniques: Control, Flow Cytometry, Activation Assay

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) Staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction for CD8, granzyme B (GZMB), CD3, Pan-CK, and DAPI. ( B ) RNA FISH against IFN-γ with staining for CD3, Pan-CK, and DAPI on mammary tissue from WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction. ( C – G ) Quantification of total CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells, CD3 + IFN-γ + cells, and Ly6G + cells in WT ( n = 5), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 6), and Stat3 –/– Chi3l1 OE ( n = 9) MIC mammary glands at 2 weeks after induction. ( H ) Staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC mammary glands at 2 weeks after induction using antibodies against Ly6G, MPO, NE, pan-CK, and DAPI. ( I and J ) Quantification of total Ly6G + MPO + cells and Ly6G + MPO + NE + cells in WT ( n = 5), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 6), and Stat3 –/– Chi3l1 OE ( n = 9) MIC mammary glands at 2 weeks after induction. ( K ) Quantification of CitH3 immunoblots normalized to α-tubulin. ( L ) Immunoblots for CitH3 and α-tubulin on WT ( n = 3), Chi3l1 OE ( n = 3), Stat3 –/– ( n = 3), and Stat3 –/– Chi3l1 OE ( n = 3) MIC mammary glands at 2 weeks after induction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test. Scale bars: 100 μm.

Article Snippet: To deplete neutrophils, cohorts of MTB MIC CHI3L1 OE mice were randomized to receive 400 μg of purified anti-LY6G rat antibody (1A8, Bio X Cell BE0075-1) or 400 μg of IgG2a rat isotype control (2A3, Bio X Cell BE0089) via intraperitoneal (i.p.) injections twice weekly.

Techniques: Staining, Western Blot

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) H&E staining of mammary glands from IgG2a- and anti-Ly6G–treated MIC Stat3 –/– Chi3l1 OE mice at 2 weeks after induction. ( B ) Quantification of hyperplastic area in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mice. Represented as percentage of mammary gland area. ( C ) Staining of mammary tissue from MIC Stat3 –/– Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for GZMB, CD8, CD3, pan-CK, and DAPI. ( D – F ) Quantification of CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mammary glands. ( G ) Staining of mammary tissue from MIC Stat3 –/– Chi3l1 OE mice treated with anti-Ly6G or IgG2a, for PD-1, CD4, CD3, pan-CK, and DAPI. ( H – J ) Quantification of CD3 + CD4 + , CD3 + CD4 + PD-1 + , and tumor-infiltrating CD3 + CD4 + T cells in IgG2a-treated ( n = 8) and anti-Ly6G–treated ( n = 8) MIC Stat3 –/– Chi3l1 OE mammary glands. ( K ) H&E staining of mammary glands from IgG2a- and anti-Ly6G–treated MIC Chi3l1 OE mice at 2 weeks after induction. ( L ) Quantification of hyperplastic area in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mice. Represented as percentage of mammary gland area. ( M ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for GZMB, CD8, CD3, pan-CK, and DAPI. ( N – P ) Quantification of CD3 + CD8 + , CD3 + CD8 + GZMB + , and tumor-infiltrating CD3 + CD8 + T cells in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. ( Q ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for 2 weeks, for PD-1, CD4, CD3, pan-CK, and DAPI. ( R – T ) Quantification of CD3 + CD4 + , CD3 + CD4 + PD1 + , and tumor-infiltrating CD3 + CD4 + T cells in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t test. Scale bars: 100 μm in A , C , G , M , and Q ; 500 μm in K .

Article Snippet: To deplete neutrophils, cohorts of MTB MIC CHI3L1 OE mice were randomized to receive 400 μg of purified anti-LY6G rat antibody (1A8, Bio X Cell BE0075-1) or 400 μg of IgG2a rat isotype control (2A3, Bio X Cell BE0089) via intraperitoneal (i.p.) injections twice weekly.

Techniques: Staining

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) H&E staining of WT, Chi3l1 OE, Stat3 –/– , and Stat3 –/– Chi3l1 OE MIC lungs at mammary tumor endpoint. ( B ) Percentage of WT ( n = 12), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 13), and Stat3 –/– Chi3l1 OE ( n = 11) MIC mice with pulmonary metastases. ( C ) Quantification of metastatic area (percent total lung area) in WT ( n = 12), Chi3l1 OE ( n = 8), Stat3 –/– ( n = 13), and Stat3 –/– Chi3l1 OE ( n = 11) MIC lungs. ( D ) Quantification of PyMT + cells (percent live cells in blood) of WT ( n = 4) and Chi3l1 OE ( n = 3) MIC mice. ( E ) FACS for circulating PyMT + cells in WT and Chi3l1 OE MIC blood at 2 weeks after induction. ( F ) Staining of WT and Chi3l1 OE MIC mammary glands at 2 weeks after induction for laminin, collagen IV, pan-CK, and DAPI. ( G and H ) Quantification of area occupied by laminin or collagen IV (percent total mammary gland area) in WT ( n = 7) and Chi3l1 OE ( n = 8) MIC mammary glands. ( I ) FACS for circulating PyMT + cells in blood of Chi3l1 OE MIC mice after anti-Ly6G or IgG2a treatment for 2 weeks. ( J ) Quantification of PyMT + cells (percent live cells in the blood) of Chi3l1 OE MIC mice treated with IgG2a ( n = 6) or anti-Ly6G ( n = 5). ( K ) Staining of mammary tissue from MIC Chi3l1 OE mice treated with anti-Ly6G or IgG2a for laminin, collagen IV, pan-CK, and DAPI. ( L and M ) Quantification of area occupied by laminin and collagen IV (percent mammary gland area) in IgG2a-treated ( n = 6) and anti-Ly6G–treated ( n = 8) MIC Chi3l1 OE mammary glands. ( N ) Schematic of Transwell invasion assay through synthetic ECM. Created in BioRender (Muller W, 2026, https://BioRender.com/4lhz2a3 ). ( O ) ECM-invading PyMT + cells with PBS, rmChi3l1, or murine neutrophils, stained with crystal violet. Neutrophils + PBS (no PyMT cells) is shown as negative control. ( P ) Quantification of invading PyMT + cells. n = 3 technical replicates for each condition. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test ( C and P ) or by unpaired Student’s t test (for D , G , H , J , L , and M ). Scale bars: 1 cm in A , left panels; 1 mm in A , right panels; 100 μm in F , K , and O .

Article Snippet: To deplete neutrophils, cohorts of MTB MIC CHI3L1 OE mice were randomized to receive 400 μg of purified anti-LY6G rat antibody (1A8, Bio X Cell BE0075-1) or 400 μg of IgG2a rat isotype control (2A3, Bio X Cell BE0089) via intraperitoneal (i.p.) injections twice weekly.

Techniques: Staining, Transwell Invasion Assay, Negative Control

Journal: JCI Insight

Article Title: The CHI3L1-neutrophil axis drives immune suppression and breast cancer metastatic dissemination

doi: 10.1172/jci.insight.199307

Figure Lengend Snippet: ( A ) Immunoblots for Chi3l1, Stat3, and α-tubulin on WT ( n = 7) and Chi3l1 –/– ( n = 6) MIC mammary endpoint tumors. ( B ) Representative H&E staining of WT and Chi3l1 –/– MIC lungs at mammary tumor endpoint. ( C ) Percentage of WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC mice with pulmonary metastases. ( D ) Quantification of total pulmonary metastatic area in WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC lungs. Represented as percentage of total lung area. ( E ) Number of lung metastatic lesions in WT ( n = 11) and Chi3l1 –/– ( n = 11) MIC lungs. ( F ) Staining of WT and Chi3l1 –/– MIC mammary glands at 2 weeks after induction, for Ly6G, MPO, NE, pan-CK, and DAPI. ( G and H ) Quantification of Ly6G + cells and Ly6G + MPO + NE + cells in WT ( n = 7) and Chi3l1 –/– ( n = 10) MIC mammary glands at 2 weeks after induction. ( I ) Staining of WT and Chi3l1 –/– MIC mammary glands at 2 weeks after induction, for laminin, collagen IV, pan-CK, and DAPI. ( J and K ) Quantification of area occupied by laminin or collagen IV in WT ( n = 7) and Chi3l1 –/– ( n = 10) MIC mammary glands at 2 weeks after induction. Represented as percentage of total mammary gland area. ( L ) FACS sorting for PyMT + tumor cells in the blood of WT MIC mice treated with anti-Chi3l1 neutralizing antibody or IgG2b isotype control at 2 weeks after induction. ( M ) Quantification of PyMT + cells as percentage of total live cells in the blood of WT MIC mice treated with IgG2b ( n = 5) or anti-Chi3l1 ( n = 5) for 2 weeks. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t test. Scale bars: 1 mm in B , left panels; 100 μm in B , right panels; 10 μm in F and I .

Article Snippet: To deplete neutrophils, cohorts of MTB MIC CHI3L1 OE mice were randomized to receive 400 μg of purified anti-LY6G rat antibody (1A8, Bio X Cell BE0075-1) or 400 μg of IgG2a rat isotype control (2A3, Bio X Cell BE0089) via intraperitoneal (i.p.) injections twice weekly.

Techniques: Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction

doi: 10.64898/2026.01.15.699253

Figure Lengend Snippet: Targeting Ly6G modulates the immune cell landscape in the heart after MI with increased pro-fibrotic γδ T cells. a) UMAP visualization of total heart CD45 + cells 5 days post MI, in mice with or without Ly6G targeting (see ). b) Cell count per mg of heart tissue for the indicated cell type clusters identified in (a). c) Macrophage content in the infarct area 14 days post-MI in isotype or anti-Ly6G treated groups. Left panel: immunofluorescence staining of CD68 (pink) on heart sections, scale bar = 200µm. Right panel: quantification of macrophage area expressed as percentage of the infarcted area. d) CellChat analysis of γδ T cells communication with cardiac fibroblasts, probability of communication pathways (ligand-receptor pairs) e) Scratch assay of 3T3 fibroblasts incubated 24h with activated γδ T cells, representative images at t=0, t=18h and t=24h (left panel) and quantification of percentage of wound closure after 18h and 24h (right panel). Each point represents one well, pooled from three independent experiments. f) Proposed model and summary of the main findings. Bar plots: one circle represents one mouse, open circles represent female mice. MI: myocardial infarction, CTL: control, Macro: Macrophages, Prolif: Proliferating, Neutro: Neutrophils, DC: Dendritic Cells, cDC1: conventional Dendritic Cell 1, DC: Dendritic Cells, pDC: plasmacytoid Dendritic Cells, NI: non-identified. Statistical tests: (panel b,c,e) unpaired t-test (*<0.05; **<0.01). Illustration (panel f) created with Biorender.

Article Snippet: For Ly6G targeting, mice received daily intraperitoneal injections of 25μg of rat IgG2aκ anti-mouse Ly6G InVivoPlusTM (BioXCell, BP0075-1) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (anti-Ly6G group), and the control group received 25μg of rat IgG2aκ isotype control (BioXCell, BP0089) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (isotype group).

Techniques: Cell Characterization, Immunofluorescence, Staining, Wound Healing Assay, Incubation, Control

Journal: bioRxiv

Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction

doi: 10.64898/2026.01.15.699253

Figure Lengend Snippet: a) UMAP plot visualization of heart neutrophils extracted from and reclustered. b) UMAP plot from panel a) split by timepoint after MI. c) Percentage of heart neutrophil clusters in total heart neutrophils according to time post MI. One circle represents one mouse, open circles represent female mice. d) Relative expression of Csf2ra , Tgfbr1 and Tgfbr2 transcripts in total neutrophils in the indicated conditions. e) relative gene expression in neutrophils left untreated or GM-CSF+TGFβ stimulated neutrophils assessed by quantitative PCR (n= neutrophils from 2 mice). f) CITE-seq signal of the indicated surface protein on bone marrow, spleen, blood and heart neutrophils at day 4 post-MI projected onto the UMAP plot from . g) Surface protein expression of the indicated markers on pre-gated live Ly6G+ neutrophils assessed by flow cytometry, in unstimulated bone marrow-isolated neutrophil, and GM-CSF+TGFβ stimulated neutrophils. MI: myocardial infarction, BM: bone marrow, CTL: control. Statistical tests: (panel c) unpaired t-test (*<0.05; **<0.01; ***<0.001; ****<0.0001).

Article Snippet: For Ly6G targeting, mice received daily intraperitoneal injections of 25μg of rat IgG2aκ anti-mouse Ly6G InVivoPlusTM (BioXCell, BP0075-1) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (anti-Ly6G group), and the control group received 25μg of rat IgG2aκ isotype control (BioXCell, BP0089) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (isotype group).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Isolation, Control

Journal: bioRxiv

Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction

doi: 10.64898/2026.01.15.699253

Figure Lengend Snippet: a) Experimental design of Ly6G targeting experiments. b) Flow cytometry analysis of heart neutrophils 3 days post MI: percentage of Neutrophils among total CD45 + cells (left panel) and percentage of SiglecF + neutrophils among total neutrophils (right panel). c) Interstitial fibrosis in the infarct border zone 14 days post-MI, expressed as percentage of total area, determined by picrosirius red staining, scale bar: 100µm. d) Experimental design of the single cell RNA-seq/CITE-seq experiments with Ly6G targeting. Group isotype MI n= 4 females and 6 males, group anti-Ly6G MI n= 5 females and 10 males, group isotype sham n= 3 males and 3 females, group anti-Ly6G sham n= 3 males and 3 females. e) UMAP plot of neutrophils identified in (Supplementary Figure 4a), extracted from the MI groups and reclustered. N=3 independent scRNA-seq/CITE-seq experiments were integrated. f) Relative expression of the main marker genes used to identify neutrophil subsets. g) Neutrophil counts per mg of heart tissue at day 5 post-MI. h) UMAP plot of neutrophils from panel e) split by treatment. i) Neutrophil cluster proportions expressed as percentage of total neutrophils. j) Gene count for 5 of the top16 up regulated biological processes in the Mature Retnlg hi cluster vs. the Mature cluster (green bars) and in the SiglecF + Ly6G lo cluster vs. the all SiglecF+ neutrophil clusters (pink bars); all with adjusted p-value <0.05 k) Expression of the indicated scores by neutrophil clusters from panel e). Bar plots: one circle represents one mouse, open circles represent female mice. MI: myocardial infarction, BM: bone marrow, Iso: isotype, GO: gene ontology. Statistical tests: (panel b, c, g, i) unpaired t-test (*<0.05; **<0.01; ***<0.001; ****<0.0001). Illustration (panel a,d) created with Biorender.

Article Snippet: For Ly6G targeting, mice received daily intraperitoneal injections of 25μg of rat IgG2aκ anti-mouse Ly6G InVivoPlusTM (BioXCell, BP0075-1) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (anti-Ly6G group), and the control group received 25μg of rat IgG2aκ isotype control (BioXCell, BP0089) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (isotype group).

Techniques: Flow Cytometry, Staining, RNA Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction

doi: 10.64898/2026.01.15.699253

Figure Lengend Snippet: a) Expression of the indicated scores in control and day 5, 7 and 14 post-MI heart cells from an in-house scRNA-seq atlas. b) Immunofluorescent staining of day 3 post-MI heart section showing the infarct and border zone area. Hoechst is stained in blue, Ly6G (neutrophils) in green and PDGFRα (fibroblasts) in red, scale bar: 50µm. c) Immunofluorescence staining of PDGFRα in the peri infarct region 3 days post-MI, expressed as PDGFRα + area percentage of total area (left panel). PDGFRα is stained in red and Hoechst in blue (right panel), scale bar: 100µm. d) Scratch assay on 3T3 fibroblasts incubated 24h with untreated or SiglecF + neutrophils; representative images at t=0 and t=24h (left panel), quantification of percentage of wound closure after 24h (right panel); each point represents one well, pooled from three independent experiments. e,f) CellChat analysis of heart neutrophil cluster (from ) communication to cardiac fibroblasts; e) Probability of communication pathways (ligand-receptor pairs); f) associated CypA and TGFβ signaling pathway network. g,h) Percentage of wound closure after 18h and 24h from scratch assay of 3T3 fibroblasts incubated 24h with SiglecF + neutrophils and Galunisertib g) or CypA inhibitor h). Each point represents one well, pooled from two independent experiments. MI: myocardial infarction, Fibro: Fibroblasts, Myo Fibro: Myofibroblatsts, VSMC: Vascular smooth muscle cell, EC: Endothelial cell, Macro: Macrophage, cDC: conventional dendritic cell, pDC: plasmacytoid dendritic cell, Neutro: Neutrophil, Mast: Mastocyte, Baso: Basophil, T: T cell, gdT: gamma delta T cell, NK: Natural Killer, ILC: innate lymphoid cell, ECM: extracellular matrix, Glycoprot: glycoproteins, prot: proteins. Statistical tests: (panel c) unpaired t-test, (panel d,g,h) Ordinary one-way ANOVA (Fisher’s LSD test) (*<0.05; **<0.01; ***<0.001).

Article Snippet: For Ly6G targeting, mice received daily intraperitoneal injections of 25μg of rat IgG2aκ anti-mouse Ly6G InVivoPlusTM (BioXCell, BP0075-1) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (anti-Ly6G group), and the control group received 25μg of rat IgG2aκ isotype control (BioXCell, BP0089) + 50μg of mouse anti-rat IgG2aκ (BioXCell, BE0122) (isotype group).

Techniques: Expressing, Control, Staining, Immunofluorescence, Wound Healing Assay, Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATG16L1 Regulates Reparative Function of Peritoneal Macrophages During Acute Drug-induced Liver Injury

doi: 10.1016/j.jcmgh.2025.101674

Figure Lengend Snippet: Intrahepatic migration of PMs promotes liver repair post-DILI. Liver tissues from APAP-treated mice were collected at 24 hours post DILI. F4/80 and GATA6 co-staining was analyzed by IHC ( A ) and IF ( B ). PMs were isolated and intraperitoneally injected into APAP-pretreated mice or depleted prior to APAP-induced DILI. Liver tissues and serum were collected at 24, 48, and 72 hours post DILI. Representative H&E ( C ), TUNEL ( D ), CD31 ( E ) staining, serum ALT ( F ) and AST ( G ), IL-1β ( H–I ), and IL-10 ( J–K ) levels were analyzed; furthermore, representative IF staining of CD11b ( L ) and Ly6G ( M ) was analyzed. n = 6/group. Data are shown as mean ± SEM. Each point represents an independent experiment. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001.

Article Snippet: For immunohistochemical (IHC) and immunofluorescence (IF) staining, the sections were dehydrated and antigens were retrieved by incubation overnight at 4°C with the following primary antibodies: rat anti-mouse F4/80 (1:100, ab11101, Abcam), mouse anti-mouse GATA6 (1:200, 67943-1-Ig, proteintech), rabbit anti-mouse ATG16L1 (1:200, ab188642, Abcam), rabbit anti-mouse platelet endothelial cell adhesion molecule-1 (CD31) (1:100, ab222783, Abcam), hyaluronan-binding protein 1/complement component 1, q subcomponent binding protein (HABP1/C1QBP) (HY- P76185 , MCE), Mouse (His) (1:100, ab213204, Abcam), rabbit anti-mouse LC3 (1:200, ab192890, Abcam), rat anti-mouse CD11b (1:50, ab8878, Abcam), rat anti-mouse Ly6G (1:500, 88876, CST), rat anti-mouse Clec4f (MAB2784; 1/1000, R&D Systems), and rabbit anti-mouse PCNA (1:200, ab29, Abcam) antibodies.

Techniques: Migration, Staining, Isolation, Injection, TUNEL Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATG16L1 Regulates Reparative Function of Peritoneal Macrophages During Acute Drug-induced Liver Injury

doi: 10.1016/j.jcmgh.2025.101674

Figure Lengend Snippet: ATG16L1 influences the pro-repair effects of PMs. Liver tissues from APAP-treated mice were collected at 24 hours post DILI. Immunostaining for F4/80, GATA6, ATG16L1, and DAPI ( A ). CD45.2 PMs were injected intraperitoneally into CD45.1 mice that had been pretreated with APAP. After 24 hours, the intrahepatic NPCs were collected. To isolate the migrated CD45.2 macrophages specifically, they were purified from the liver sample using CD45.2-based magnetic sorting. Stable CD45.2 PMs were used as the control group. The samples were analyzed by flow cytometry ( B ) and WB for ATG16L1 ( C ); n = 3/group. WB analysis of ATG16L1 in peritoneal macrophages ( D ). PMs were injected into APAP-pretreated WT mice, and liver tissues/serum were collected at 0, 24, 48, and 72 hours post DILI. H&E ( E ), TUNEL ( F ), CD31 ( G ), ALT ( H ), AST ( I ), IL-1β ( J–K ), and IL-10 ( L–M ) analyses. Furthermore, representative IF staining of CD11b ( N ) and Ly6G ( O ) was evaluated. n = 6/group. Data are shown as mean ± SEM. Each point represents an independent experiment. ∗ P < .05, ∗∗ P < .01, ∗∗∗∗ P < .0001.

Article Snippet: For immunohistochemical (IHC) and immunofluorescence (IF) staining, the sections were dehydrated and antigens were retrieved by incubation overnight at 4°C with the following primary antibodies: rat anti-mouse F4/80 (1:100, ab11101, Abcam), mouse anti-mouse GATA6 (1:200, 67943-1-Ig, proteintech), rabbit anti-mouse ATG16L1 (1:200, ab188642, Abcam), rabbit anti-mouse platelet endothelial cell adhesion molecule-1 (CD31) (1:100, ab222783, Abcam), hyaluronan-binding protein 1/complement component 1, q subcomponent binding protein (HABP1/C1QBP) (HY- P76185 , MCE), Mouse (His) (1:100, ab213204, Abcam), rabbit anti-mouse LC3 (1:200, ab192890, Abcam), rat anti-mouse CD11b (1:50, ab8878, Abcam), rat anti-mouse Ly6G (1:500, 88876, CST), rat anti-mouse Clec4f (MAB2784; 1/1000, R&D Systems), and rabbit anti-mouse PCNA (1:200, ab29, Abcam) antibodies.

Techniques: Immunostaining, Injection, Purification, Control, Flow Cytometry, TUNEL Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Neutrophils are recruited to lungs of both C57BL/6 and C3HeB/FeJ mice, while macrophage and CD4 + T cell accumulation is delayed in TB-susceptible C3HeB/FeJ mice. (a–c) C57BL/6 and C3HeB/FeJ mice were aerosol infected with (a) HN878, (b) 6C4, or (c) 4I2 M. tuberculosis strains, and numbers of neutrophils (Ly6G hi CD11b hi CD45 + ), total and MHC-II + MDMs (HN878: Siglec F − Ly6G − CD11b + CD64 + MerTK + CD45 + ; 6C4/4I2: Siglec F − Ly6G − CD11b + F4/80 + CD45 + ), and CD44 + CD62L − CD4 + T cells (CD3ε + CD45 + ) in lung tissue were determined by flow cytometry. Flow cytometry gating was performed as represented in . Points show individual replicate mice with lines at the mean. Statistical testing: two-way ANOVA with Holm–Sidak post hoc test; actual adjusted P value are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Data shown are from a single experiment per M. tuberculosis strain with N = 3–5 mice per group and are representative of a minimum of two independent experiments. See also .

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Aerosol, Infection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Spatial separation of CD4 + T cells and neutrophils in TB lesions. C57BL/6 (B6) and C3HeB/FeJ (C3H) mice were aerosol infected with M. tuberculosis HN878 and lungs harvested for multiparameter immunofluorescence staining of formaldehyde-fixed, paraffin-embedded sections at the indicated time points. (a) Representative low-power images of lung lobes, showing distribution of lesions. White boxes indicate areas at days 14 and 21 shown at greater magnification in panel d. The images from days 14 and 21 are reproduced in , along with those from the other replicate mice at these time points. Scale bars = 1 mm. (b) Quantification of numbers of lesions in whole lungs, normalized for tissue area. (c) Median area of lesions detected per mouse. (d) Representative images of lesions at 14 and 21 days after infection, showing all immune cell markers. Images are shown at different scales to aid visualization (day 14 scale = 20 µm; day 21 scale = 50 µm). (e) Number of cells positive for the indicated markers within lesions, normalized for the total area of all lesions. (f) Numbers of CD4 + T cells in contact with a macrophage annotation (≤0 μm distance), normalized for the total area of all lesions. (g) Stacked bar plots showing percentages of lesions across whole lungs falling into low (≤20%), intermediate (Int, >20% ≤40%), or high (Hi, >40) bins for coverage with Ly6G staining. Data shown are means ± standard error of all mice with detectable lesions ( N = 3 for day 14 C3HeB/FeJ; N = 4 for others). (h) Representative images showing the relative distribution of CD4 + T cells and Ly6G staining in lesions with low and high Ly6G coverage. Scale bar = 50 μm. (i) Number of CD4 + T cells within lesions in the different Ly6G coverage bins at 21 days after infection, normalized for the total area of lesions analyzed per bin. Plots in b, c, e, f, and i show points representing all individual replicate mice with detectable lesions, with lines at the mean. Data shown are from a single experiment with N = 4 mice per group and are representative of two independent experiments. Statistical analysis: b, c, and e, two-way ANOVA with Holm–Sidak post hoc test; f, unpaired t test; g, Dirichlet-multinomial regression, with the indicated P values corresponding to the mouse strain effect on frequency of Ly6G-high lesions; i, one-way ANOVA with Holm–Sidak post hoc test. Actual adjusted P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also: .

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Aerosol, Infection, Immunofluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Neutrophil depletion increases macrophage activation and CD4 + T cell numbers in lung lesions in TB-susceptible C3HeB/FeJ mice. (a) C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878 and received intraperitoneal injection of either anti-Ly6G (αLy6G) or isotype control three times per week between days 12 and 25. Tissues were analyzed at 20 and 26 days after infection. (b) Representative images of S100A9 immunohistochemistry in lung sections at 20 days after infection, confirming neutrophil depletion in αLy6G-treated mice. Scale bars = 100 μm. (c) Lung M. tuberculosis CFU counts. (d) Numbers of total, Ly6C + MHC-II − and MHC-II + MDMs (Siglec F − Ly6G − CD11b + CD64 + MerTK + CD45 + ) in lung tissue as determined by flow cytometry. (e) Numbers of CD44 + CD62 − CD4 + T cells (CD3ε + CD45 + ) in lung tissue as determined by flow cytometry. (f) Representative images of lung lesions showing macrophage (CD68, magenta), CD4 + T cells (CD4, green), and neutrophil (Ly6G, white) staining at 20 and 26 days after infection. Scale bars = 100 μm. (g) Number of CD4 + T cells within lung lesions, normalized for the total area of all lesions across whole left lungs. (h) Numbers of CD4 + T cells in contact with a macrophage annotation (≤0 μm distance), normalized for the total area of all lesions across whole left lungs. Points show individual replicate mice with lines at the mean. Data shown are from a single experiment with N = 5 mice per group, representative of two independent experiments. Statistical analysis: (c–e) two-way ANOVA with Holm–Sidak post hoc test; (g and h) unpaired t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See also: .

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Activation Assay, Aerosol, Infection, Injection, Control, Immunohistochemistry, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Type I IFN-inducible gene expression and NETs in lungs of C57BL/6 and C3HeB/FeJ mice. (a) Dot plots showing expression of individual representative ISGs or a 37-gene type I IFN-response signature in myeloid cell populations in scRNA-seq data. (b–g) Analysis of lung multiplex immunofluorescence in HN878-infected mice with and without IFNAR blockade in the experiments described in and . (b and c) Numbers of total CD4 + T cells in whole left lungs normalized for tissue area. (d) Abundance of CD4 + T cells in lung lesions expressed relative to that in non-lesional lung. Statistical analysis: unpaired t test, with Welch’s correction applied in b. (e) Representative images showing all merged channels for NET staining (top) and CitH3 and DAPI alone (bottom) in C3HeB/FeJ mice at 20 days after infection. Scale bar = 50 μm. (f) Quantification of CitH3 NET staining relative to Ly6G staining in lung lesions. Statistical analysis: unpaired t test. (g) Percentage of lung lesions with low (CitH3/Ly6G < 0.2), intermediate (CitH3/Ly6G 0.2–0.4), or high (CitH3/Ly6G > 0.4) NET burden. Data shown are means ± standard error. Statistical analysis shown is Dirichlet-multinomial regression analysis of the effect of αIFNAR treatment on lesion NET status. Symbols indicate significant differences in the proportion of NET-low lesions. Data in b–g are from single experiments with N = 5 mice per group and are representative of two independent experiments. (h) DESeq2-normalized expression values of genes of interest as identified in in whole lungs early in infection with HN878. Data shown are from a single bulk RNA-seq experiment with N = 5 mice per group and represent individual replicate mice as points with lines at the mean. Statistical analysis: two-way ANOVA with Holm–Sidak post hoc test. (i) Dot plot showing expression of Slfn4 in scRNA-seq clusters. Data in a and i are from a single scRNA-seq experiment, and plots show combined data from cells from N = 3 mice per group. Circle sizes represent the abundance of cells expressing the gene, as a percentage of either cells in the cluster (a) or within total cells (i). Circle color is proportional to the mean expression of the gene within all cells in the cluster. Actual P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Gene Expression, Expressing, Multiplex Assay, Immunofluorescence, Infection, Staining, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Type I IFN signaling impairs early M. tuberculosis control in both C57BL/6 and highly TB-susceptible C3HeB/FeJ mice. (a) C57BL/6 mice were aerosol infected with M. tuberculosis HN878 and received intraperitoneal injection of either anti-IFNAR (αIFNAR) or isotype control three times per week either between days −1 and 13 (†) or days −1 and 27 (‡). (b and c) CFU counts in lung tissue from either the (b) early (†) or (c) continuous (‡) αIFNAR treatment regimen. (d) Numbers of neutrophils (Ly6G hi CD11b hi CD45 + ), total and Ly6C − MHC-II + MDMs (Siglec F − Ly6G − CD11b + CD64 + MerTK + CD45 + ), and CD44 + CD62 − CD4 + T cells (CD3ε + CD45 + ) in lung tissue, as determined by flow cytometry. (e) C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878 and received intraperitoneal injection of either αIFNAR or isotype control three times per week between days −1 and 18. (f) CFU counts in lung tissue. (g) Numbers of neutrophils, total and Ly6C − MHC-II + MDMs, and CD44 + CD62 − CD4 + T cells in lung tissue, as determined by flow cytometry. Points represent individual replicate mice with lines at the mean. Data are from single experiments with N = 4–5 mice per group and are representative of two independent experiments. Statistical analysis for CFU counts at day 26–28: unpaired t test. All other statistical analysis: two-way ANOVA with Holm–Sidak post hoc test. Actual adjusted P values are shown or: *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Control, Aerosol, Infection, Injection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Type I IFN drives neutrophil swarming, impeding lung T cell–macrophage interactions and TB control

doi: 10.1084/jem.20250466

Figure Lengend Snippet: Early type I IFN signaling promotes neutrophil swarming and limits CD4 + T cell numbers in TB lesions of both relatively resistant and highly TB-susceptible mice. Lung sections from experiments described in were analyzed by multiparameter immunofluorescence. (a) Images of representative lesions from C57BL/6 mice treated with either early anti-IFNAR (αIFNAR) or isotype control, at 20 days after infection. Individual fluorescent channels and merged images are shown. (b) Stacked bar plots showing percentages of lesions across whole left lungs falling into low (≤20%), intermediate (Int, >20% ≤40%), or high (Hi, >40) bins for coverage with Ly6G staining. (c and d) Numbers of total CD4 + T cells (c) and CD4 + T cells in contact with a macrophage annotation (≤0 μm distance, d) within lung lesions at 20 days after infection, normalized for the total area of all lesions across whole left lungs. (e) Images of representative lesions from C3HeB/FeJ mice treated with either early anti-IFNAR (αIFNAR) or isotype control at 26 days after infection. Individual fluorescent channels and merged images are shown. (f) Stacked bar plots showing percentages of lesions across whole left lungs falling into low (≤20%), intermediate (Int, >20% ≤40%), or high (Hi, >40) bins for coverage with Ly6G staining. (g and h) Number of total CD4 + T cells (g) and CD4 + T cells in contact with a macrophage annotation (≤0 μm distance, h) within lung lesions at the indicated time points, normalized for the total area of all lesions across whole left lungs. (i–l) Immunofluorescence staining for CitH3 and Ly6G to detect NETs in lung lesions at the indicated time points. (i and k) Representative images showing all merged channels (top) or CitH3 and DAPI alone (bottom) in C3HeB/FeJ (i) and C57BL/6 (k) mice. (j and l) Quantification of CitH3 NET staining relative to Ly6G staining in lung lesions. Data shown in b and f are means ± standard error of N = 5 per group. Statistical analysis shown in b and f is Dirichlet-multinomial regression analysis of the effect of αIFNAR treatment on lesion composition. Symbols indicate significant differences in the proportion of Ly6G-low lesions. Plots in c, d, g, h, j, and l show individual replicate mice as points with lines at the mean or median (h). Statistical analysis in b and g: unpaired t test. Statistical analysis in h: Mann–Whitney test. Statistical analysis in j and l: unpaired t test with Welch’s correction. Actual P values are shown or: *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. Data shown are from single experiments with N = 5 mice per group that are representative of two independent experiments. Scale bars in a and e = 100 μm; scale bars in i and k = 50 μm. See also: .

Article Snippet: For neutrophil depletion, mice received 0.2 mg of rat anti-mouse Ly6G (clone 1A8; Bio X Cell) or rat IgG 2a isotype control (clone 2A3; Bio X Cell) in 0.2 ml volume of sterile PBS by intraperitoneal injection.

Techniques: Immunofluorescence, Control, Infection, Staining, MANN-WHITNEY