fitc ly6c  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: β-Glucan Reprograms Neutrophils to Induce Disease Tolerance Against Influenza A Virus

doi: 10.1101/2024.09.02.610822

Figure Lengend Snippet: (A-D) Mice were treated with β--glucan 6 days before influenza infection. On day 9 post-glucan treatment, blood (A-D) and Lung (E-H) neutrophils from infected and non-infected mice were extracted and their surface phenotype was assessed by spectral flow cytometry. (A) Uniform Manifold Approximation and Projection (UMAP) of CD11b+ Ly6G+ neutrophils from control and β--glucan treated mice with and without influenza infection. Neutrophils separate in two main Flowsom Clusters (cluster 1, grey and cluster 2, Cyan, 96% of neutrophils) (B) UMAP from A is projected for the 4 experimental group (PBS +/- Influenza, black and β--glucan +/- Influenza, Dark Blue). Neutrophil density repartition shows a shift of neutrophils in β-glucan treated group from Cluster 1 to Cluster 2. (C) Quantification of neutrophil repartition. (D) MFIs of selected markers are projected on the UMAP from (A). Histograms show MFIs from Cluster 1 (grey and cluster 2 (Cyan). Cluster 2 neutrophils exhibit lower expression of classical maturation markers CD101, Ly6G and CXCR2 and present with a less activated phenotype (CD62Lhigh, CD11blow, CD49dlow). (E) UMAP of CD11b+ Ly6G+ neutrophils from control and β--glucan treated mice with and without influenza infection. Neutrophils separate in 8 Flowsom Clusters. (F)UMAP from A is projected for the 4 experimental group (PBS +/- Influenza, black and β-glucan +/- Influenza, Dark Blue). Clusters 5, 6, and 8 expand dramatically during influenza infection. (G) Quantification of neutrophil present in influenza-driven clusters (Clusters 5, 6, 8). (H) MFIs of selected markers are projected on the UMAP from (E). Influenza-driven clusters exhibit an activated phenotype (CD14high, CD24high CD11b high, CD62Llow, CD49dlow). MFI Mean Fluorescence Intensity. UMAPs are based on 16 surface markers (CD45, Ly6G, CD101, CXCR2, CXCR4, CD62L, CD24, CD11b, CD49d, CD44, CCR2, Ly6C, CD80, MHCII, CD16, CD14). n=7. (I-O) Mice were treated with β-glucan. Neutrophils were purified from blood on day 4 post- glucan treatment. Neutrophils’ cellular metabolism was determined by seahorse (J), basal respiration (K), maximal respiration (L), and ATP (M) production are shown. (N, O) Representative histogram plot and quantification (MFI) for mitochondrial mass using mitotracker green dye in the neutrophils of β-glucan-treated mice. (P) Representative 3-dimensional reconstructed lung intravital microscopy images from control or β-glucan treated Ly6G-TdTom mice. Mitotracker green dye was given i.v. prior to imaging. (Q) Mitochondria bright neutrophils were quantified. All scale bars = 50µm. (R-U) Ifnar1 -/- mice were treated with β-glucan. Neutrophils were purified from blood on day 4 post β-glucan treatment. Neutrophils’ cellular metabolism was determined by seahorse (R) and basal respiration (S). (T, U) Representative histogram plot (T) and quantification (MFI) for mitochondrial mass (U) using mitotracker green dye in the neutrophils from the lungs of β-glucan-treated Ifnar1 -/- mice. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons tests and unpaired t-test. * p<0.05, ** p <0.01, ***p<0.001.

Article Snippet: Antibodies for the innate panel: anti-CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), Siglec-F–PE-CF594 (clone E50– 2440, BD Bioscience), F4/80–APC (clone BM8, eBioscience), Ly6C–FITC (clone AL-21, BD Bioscience), Ly6G–PerCP-eFluor710 (clone 1A8, eBioscience).

Techniques: Infection, Flow Cytometry, Control, Expressing, Fluorescence, Purification, Intravital Microscopy, Imaging

Journal: bioRxiv

Article Title: β-Glucan Reprograms Neutrophils to Induce Disease Tolerance Against Influenza A Virus

doi: 10.1101/2024.09.02.610822

Figure Lengend Snippet: (A-D) Mice were treated with β--glucan 6 days before influenza infection. On day 9 post-glucan treatment, blood (A-D) and Lung (E-H) neutrophils from infected and non-infected mice were extracted and their surface phenotype was assessed by spectral flow cytometry. (A) Uniform Manifold Approximation and Projection (UMAP) of CD11b+ Ly6G+ neutrophils from control and β--glucan treated mice with and without influenza infection. Neutrophils separate in two main Flowsom Clusters (cluster 1, grey and cluster 2, Cyan, 96% of neutrophils) (B) UMAP from A is projected for the 4 experimental group (PBS +/- Influenza, black and β--glucan +/- Influenza, Dark Blue). Neutrophil density repartition shows a shift of neutrophils in β-glucan treated group from Cluster 1 to Cluster 2. (C) Quantification of neutrophil repartition. (D) MFIs of selected markers are projected on the UMAP from (A). Histograms show MFIs from Cluster 1 (grey and cluster 2 (Cyan). Cluster 2 neutrophils exhibit lower expression of classical maturation markers CD101, Ly6G and CXCR2 and present with a less activated phenotype (CD62Lhigh, CD11blow, CD49dlow). (E) UMAP of CD11b+ Ly6G+ neutrophils from control and β--glucan treated mice with and without influenza infection. Neutrophils separate in 8 Flowsom Clusters. (F)UMAP from A is projected for the 4 experimental group (PBS +/- Influenza, black and β-glucan +/- Influenza, Dark Blue). Clusters 5, 6, and 8 expand dramatically during influenza infection. (G) Quantification of neutrophil present in influenza-driven clusters (Clusters 5, 6, 8). (H) MFIs of selected markers are projected on the UMAP from (E). Influenza-driven clusters exhibit an activated phenotype (CD14high, CD24high CD11b high, CD62Llow, CD49dlow). MFI Mean Fluorescence Intensity. UMAPs are based on 16 surface markers (CD45, Ly6G, CD101, CXCR2, CXCR4, CD62L, CD24, CD11b, CD49d, CD44, CCR2, Ly6C, CD80, MHCII, CD16, CD14). n=7. (I-O) Mice were treated with β-glucan. Neutrophils were purified from blood on day 4 post- glucan treatment. Neutrophils’ cellular metabolism was determined by seahorse (J), basal respiration (K), maximal respiration (L), and ATP (M) production are shown. (N, O) Representative histogram plot and quantification (MFI) for mitochondrial mass using mitotracker green dye in the neutrophils of β-glucan-treated mice. (P) Representative 3-dimensional reconstructed lung intravital microscopy images from control or β-glucan treated Ly6G-TdTom mice. Mitotracker green dye was given i.v. prior to imaging. (Q) Mitochondria bright neutrophils were quantified. All scale bars = 50µm. (R-U) Ifnar1 -/- mice were treated with β-glucan. Neutrophils were purified from blood on day 4 post β-glucan treatment. Neutrophils’ cellular metabolism was determined by seahorse (R) and basal respiration (S). (T, U) Representative histogram plot (T) and quantification (MFI) for mitochondrial mass (U) using mitotracker green dye in the neutrophils from the lungs of β-glucan-treated Ifnar1 -/- mice. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons tests and unpaired t-test. * p<0.05, ** p <0.01, ***p<0.001.

Article Snippet: Antibodies for the innate panel: anti- CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), anti-Siglec-F–PE-CF594 (clone E50–2440, BD Biosciences), anti-F4/80–APC (clone BM8, eBioscience), anti-Ly6C–FITC (clone AL-21, BD Bioscience), anti-Ly6G–PerCP– eFluor710 (clone 1A8, eBioscience).

Techniques: Infection, Flow Cytometry, Control, Expressing, Fluorescence, Purification, Intravital Microscopy, Imaging