Journal: Microbial Biotechnology

Article Title: Disarming the Pathogenicity of Methicillin‐Resistant Staphylococcus aureus via Osmundacetone‐Mediated Inhibition of Sortase A

doi: 10.1111/1751-7915.70119

Figure Lengend Snippet: In vivo therapeutic effect of OSC on mice with S. aureus ‐induced pneumonia. (a) Experimental flowchart outlining the experimental design and steps of the S. aureus ‐induced pneumonia mouse model. (b) Effects of different treatments on the survival rate of mice with pneumonia over a period of 96 h. The survival rates of the OSC‐treated group were compared with those of the control group via observation and recording. (c) Bacterial load of pneumonia‐infected mice by the serial dilution method and homogenised lung tissue plate counting. The data are expressed as the means ± SDs, and each group contained six biologically independent animals. (d) Statistical plot of the dry and wet specific gravity of the lung tissue in each group of mice, which was used to assess the extent of pulmonary oedema. (e) The proportions of neutrophils and lymphocytes in the lung tissue of the mice in each group were detected by flow cytometry. FITC‐labelled Ly6G and PE‐labelled CD11b were used to distinguish and quantify different types of immune cells. (f) Microscopic changes in mouse lung tissues were demonstrated by H&E staining. (g) Microscopic changes in mouse lung tissues were demonstrated by F4/80 staining. Histopathological changes in the lungs of the rats in the OSC (40 mg/kg)‐treated group were compared with those in the WT group, and the scale bar represents 50 μm.

Article Snippet: After centrifugation for another 10 min, the supernatant was discarded, and the cell precipitate was resuspended in blocking solution and blocked on ice for 10 min. After the blocking solution was discarded, the cells were mixed with appropriately diluted PE‐labelled anti‐mouse/human CD11b antibody (Elabscience, China) and FITC‐labelled anti‐mouse Ly6G antibody (Elabscience, China) and incubated for 30 min at 4°C in the dark.

Techniques: In Vivo, Control, Infection, Serial Dilution, Flow Cytometry, Staining

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Diagram of the procedure for intravital imaging of mouse liver. b , Intravital imaging of neutrophils in mouse liver. Neutrophils were labelled with PE–anti-Ly6G (Gr1; green) and blood vessels were labelled with AF647–WGA (purple). Scale bars, 30 μm (top) and 10 μm (magnified; bottom). The arrowheads indicate migrasomes. c , Diagram of the procedure for preparing samples for imaging flow cytometry analysis. d , Gating strategy of imaging flow cytometry analysis. Blood was stained with PE–anti-Ly6G and APC–anti-CD41. Neutrophil-derived migrasomes (R3; Ly6G + ) and platelets (R4; CD41 + ) were gated from the small particle population (R2). e , Images of neutrophil-derived migrasomes and platelets from ImageStream. Scale bars, 10 μm. f , Quantification of neutrophil-derived migrasomes (NeuMigs) and platelets (PLTs) in mouse blood by ImageStream analysis. n = 20 mice. g , h , SEM images of a CES ( g ) and a platelet ( h ). Scale bars, 1 μm. i , Measurement of the diameter of migrasomes and platelets. n = 50 particles per group. j , Diagram of the procedure for positive (top) and negative (bottom) isolation of neutrophil-derived migrasomes from blood. k , SEM images of anti-Ly6G-conjugated beads and positively sorted neutrophil-derived migrasomes (psNeuMigs). Scale bars, 1 μm. l , Flow cytometry analysis of negatively sorted neutrophil-derived migrasomes (nsNeuMigs) stained with PE–anti-Ly6G. Particles positively isolated with the kit beads served as a control (Ctrl). Samples from 20 mice were pooled and analysed together. m , SEM images of nsNeuMig incubated with anti-Ly6G-conjugated magnetic beads. Scale bars, 1 μm. n , Percentage of psNeuMig and nsNeuMig with or without retraction fibres. n = 43 for psNeuMig and n = 54 for nsNeuMig. o , nsNeuMigs and NETs were normalized with total protein and subjected to western blot analysis using antibodies against markers for NETs and migrasomes. p , Flow cytometry analysis of nsNeuMig and NMPs after staining with Annexin V-FITC. Unstained NMPs served as a negative control. q , Isolated neutrophil-derived migrasomes and microvesicles were normalized with total protein and subjected to western blot analysis using antibodies against the indicated molecules. All statistical data are presented as means ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. The western blot grey values were quantified using ImageJ. Source numerical data and unprocessed blots are available in Source Data Fig. . RMS, root mean square. BF, bright field; Sup, supernatant.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Imaging, Flow Cytometry, Staining, Derivative Assay, Isolation, Control, Incubation, Magnetic Beads, Western Blot, Negative Control, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Flow cytometry analysis of the blood white cells after staining with PE-anti-Ly6G and APC-anti-CD41 by CytoFLEX. b , The CD41 + Ly6G + population of the white cells was sorted by MoFlo-Astrios-EQ and imaged by Dragonfly confocal microscopy. Scale bar, 20 μm. c , Platelets were isolated and stained with PE-anti-Ly6G and APC-anti-CD41 for Dragonfly imaging. Scale bar, 10 μm. d , Diagram of the procedures for preparing CES from the blood of platelet-depleted mice (left), and for purifying platelets from the blood of neutrophil-depleted mice (right). e , Flow cytometry analysis of whole blood cells from control (left panels), neutrophil-depleted (middle panels), and platelet-depleted mice (right panels) after staining with PE-anti-Ly6G or APC-anti-CD41. Samples from five mice were pooled and analyzed together. f , Flow cytometry analysis of purified platelets (PLTs) and CES after staining with PE-anti-Ly6G and APC-anti-CD41. Samples from five mice were pooled and analyzed together. g-h , SEM image of CESs ( g ) or platelets ( h ). These results come from the same experiments as Fig. . Scale bar, 1 μm. i , SEM images of nsNeuMigs which were incubated with anti-Ly6G-conjugated magnetic beads. Scale bar, 10 μm. The migrasome in the dashed-box is enlarged at the left. Scale bar, 1 μm. j , SEM images of NETs. Scale bar, 5 μm (left); 1 μm (enlarged). k , Immunofluorescence staining images of migrasomes and NETs. Scale bar, 20 μm. l , SEM image of NMPs. Scale bar, 1 μm. m-n , Dragonfly confocal images of nsNeuMigs ( m ) or NMPs ( n ) stained with PE-anti-Ly6G (Gr1) and Annexin V-FITC. Scale bar, 20 μm. o . NMPs were compared with nsNeuMig by western blot analysis. The samples were normalized with total protein level. Gray values of western blots were quantified by Image J. Source unprocessed blots are available in source data.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Flow Cytometry, Staining, Confocal Microscopy, Isolation, Imaging, Control, Purification, Incubation, Magnetic Beads, Immunofluorescence, Western Blot

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Volcano plot showing the differential abundance of proteins in isolated neutrophil-derived migrasomes versus platelets. Migrasomes and platelets were subjected to label-free quantitative mass spectrometry analysis. The purple dots represent a migrasome/platelet abundance ratio of ≥2 ( P < 0.05) and the cyan dots represent a migrasome/platelet abundance ratio of ≤0.5 ( P < 0.05). n = 3 biologically independent experiments. P values were calculated using a two-tailed, unpaired t -test. b , Heat map of the distribution of coagulation factors in platelets and neutrophil-derived migrasomes. The coloured scale bars represent relative values. c , d , psNeuMigs ( c ) or nsNeuMigs ( d ) were isolated from the blood of platelet-depleted mice and platelets were isolated from the blood of neutrophil-depleted mice. S-plasma is the supernatant after centrifuging plasma at 20,000 g , 4 ℃ for 1 h. These samples were normalized with total protein and subjected to western blot analysis. e , Confocal microscopy images of purified neutrophil-derived migrasomes revealed by immunofluorescence staining using anti-Ly6G and anti-thrombin, anti-prothrombin, anti-factor X or anti-factor XIII. Scale bars, 1 μm. f , Thrombin activity assay using the internally quenched 5-FAM/QXL-520 FRET substrate of thrombin. nsNeuMigs, platelets and s-plasma were prepared and normalized with total protein, then mixed with the thrombin substrate for fluorescence detection by EnSpire microplate reader. Thrombin (0.5 U ml −1 ) served as a positive control. g , psNeuMigs, neutrophils and platelets were normalized with total protein and subjected to western blot analysis using antibodies against the indicated molecules. h , Diagram of the procedure for the digestion of purified migrasomes by proteinase K (PK) and subsequent incubation with s-plasma. i – k , CESs ( i ), platelets ( j ) or nsNeuMigs ( k ) were isolated from mice and digested with 100 μg ml −1 PK at 37 °C for 30 min, then incubated with s-plasma at 37 °C for 1 h. The samples (pre-digestion (ctrl), PK digested (PK), PK digested then s-plasma incubated (PK_s-pla) and s-plasma (s-pla)) were subjected to western blot analysis using antibodies against the indicated molecules. The grey values for the western blots were quantified using ImageJ. Source numerical data and unprocessed blots are available in Source Data Fig. . NoDiff, no difference.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Isolation, Derivative Assay, Mass Spectrometry, Two Tailed Test, Coagulation, Clinical Proteomics, Western Blot, Confocal Microscopy, Purification, Immunofluorescence, Staining, Activity Assay, Fluorescence, Positive Control, Incubation

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Western blot analysis of coagulation factors in platelets, nsNeuMig, and s-plasma. S-plasma is the supernatant after centrifuging plasma at 20,000 g for 1 hour. b , Dragonfly confocal microscopy images of nsNeuMigs revealed by immunofluorescence staining (IF). These results come from the same experiments as Fig. . Migrasomes were negatively isolated and fixed with 4% paraformaldehyde for IF using anti-Ly6G and anti-thrombin, anti-prothrombin, anti-factor X and anti-factor XIII. Scale bar, 10 μm. Western blot gray values were quantified by Image J. Source unprocessed blots are available in source data.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Western Blot, Coagulation, Clinical Proteomics, Confocal Microscopy, Immunofluorescence, Staining, Isolation

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , b , Flow cytometry analyses of platelet activation. Platelets were isolated from neutrophil-depleted mouse blood and stimulated with PBS, thrombin (1 U ml −1 ) or nsNeuMig (nsNeuMig:platelet = 1:2). Platelet activation is indicated by CD62P ( a ) and platelet morphology is indicated by SSC and FSC ( b ). c , Platelets activated by thrombin or neutrophil-derived migrasomes were stained with APC–anti-CD41 (cyan), PE–anti-CD62P (purple) and AF488–anti-Ly6G (yellow) and imaged by three-dimensional Dragonfly confocal microscopy. Scale bars, 20 μm. d , Measurement of the diameter and area of platelets (Ctrl) and platelet aggregates induced by thrombin and nsNeuMig. n = 104 platelets for Ctrl and n = 106 and n = 138 platelet aggregates for thrombin and nsNeuMig, respectively. e , SEM images of platelets activated in vitro by thrombin or nsNeuMig. Yellow arrowheads indicate migrasomes coated with anti-Ly6G-conjugated magnetic beads. Cyan arrowheads indicate platelets. Scale bar for the left three panels, 2 μm. The migrasomes and platelets in the dashed box are enlarged on the right (scale bar, 1 μm). f , Measurement of platelet protrusion length. n = 61 platelets for Ctrl, n = 64 platelets for thrombin and n = 62 platelets for nsNeuMig. g , Flow cytometry analysis of platelet activation. Platelets were isolated from neutrophil-depleted mouse blood and stimulated with PBS, thrombin or neutrophil-derived migrasomes (nsNeuMig:PLT = 1:2, 1:10, 1:20, 1:50, 1:100 or 1:300). Platelet activation is indicated by CD62P. All statistical data are presented as means ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. Source numerical data are available in Source Data Fig. .

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Flow Cytometry, Activation Assay, Isolation, Derivative Assay, Staining, Confocal Microscopy, In Vitro, Magnetic Beads, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a, b, d , Intravital imaging of non-wounded liver ( a ), wounded liver ( b ) or kidney ( d ). AF488-WGA labels blood vessels, PE-anti-Ly6G (Gr1) labels neutrophil-derived migrasomes, and APC-anti-CD41 labels platelets. Scale bar, 20 μm ( a , b ), 50 μm ( d ). The dashed-white-line indicates the wound boundary. c, e . Quantification of the ratio of platelets to neutrophil-derived migrasomes at the liver ( c ) or kidney ( e ) injury site. n = 17 mice ( c ), n = 15 field-of-views from 3 mice ( e ). f , Imaging of exogenous-injected neutrophil-migrasomes in wounded liver. Scale bar, 20 μm. g , Imaging of platelets and neutrophil-derived migrasomes within whole blood which were placed in non-coated channels. Scale bar, 20 μm. h , Quantification of the ratio of platelets to migrasomes in non-coated channels with no flow. n = 5 mice. i, l . Dragonfly images present the in vitro flow assay using ctrl and GFOGER (200 μg/mL for 30 min) ( i ) or anti-integrin α2 (HMα2) (5 μg/mL for 25 min) ( l ) treated blood. Scale bar, 10 μm. j, m . Statistical analysis of the number of neutrophil-derived migrasomes accumulated at collagen exposing site in each group. n = 35 (Ctrl) 40 (GFOGER) 55 (Ctrl) 57 (anti-integrin α2) field-of-views. k , Heat map of the distribution of adhesion molecules in platelets and neutrophil-derived migrasomes revealed by quantitative mass spectrometry analysis. n-o . Confocal images of nsNeuMigs revealed by immunofluorescence staining. Scale bar, 1 μm. p . Diagram showing how the FRET assay assess the conformation of integrins. q, s . Confocal images of neutrophils and in vitro generated migrasomes ( q ), or isolated human neutrophil-derived migrasomes, neutrophils and platelets ( s ) revealed by immunofluorescence staining using anti-integrin β1 (AIIB2-Fab), anti-CD66b and FM4-64-FX before bleach. Scale bar, 10 μm ( q ), 5 μm ( s ). r, t . FRET measurement of the samples in q and s . n = 20 for each group. All of the statistical data are presented as the mean ± s.e.m, P values were calculated using the two-tailed, unpaired t-test. Data were pooled from six ( c ), three ( e , j , k ), and four ( m ) independent experiments. Source numerical data are available in source data.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Imaging, Derivative Assay, Injection, In Vitro, Mass Spectrometry, Immunofluorescence, Staining, Generated, Isolation, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Wounded lung imaging. AF488–WGA-labelled vessels (cyan), PE–anti-Ly6G (Gr1)-labelled neutrophil-derived migrasomes (yellow) and APC–anti-CD41-labelled platelets (purple) are shown. The dashed white lines indicate the wound boundary. Scale bar, 20 μm. b . Plot showing the ratio of platelets to neutrophil-derived migrasomes at injury sites. n = 20 fields of view. c , Diagram of the flow channel used for the in vitro flow assay. d , Dragonfly images of the flow assay. Scale bars, 50 μm. The dashed line indicates the boundary of collagen coating. e , Plot showing the ratio of platelets to neutrophil-derived migrasomes at collagen-coated sites. n = 7 mice. f , Western blot analysis of platelets and nsNeuMigs. g , Tail tip bleeding assay in control, neutrophil-depleted (anti-Ly6G) and platelet-depleted (anti-CD41) mice. Bloods were dripped on a clear plastic sheet for imaging. h , Statistical analysis of blood volume. n = 14 (Ctrl), 10 (anti-Ly6G) and 13 (anti-CD41) mice. i , Tail tip bleeding assay in control, neutrophil-depleted mice and mice injected with nsNeuMig (i.v.; 2 × 10 6 per mouse). j , Statistical analysis of blood volume. n = 20 mice per group. k , Dragonfly images of in vitro flow assay using blood from control and neutrophil-depleted mice. Scale bars, 50 μm. l , Measurement of CD41 intensity in the flow channel. n = 8 (Ctrl) and 12 (anti-Ly6G) mice. m , Quantification of neutrophil-derived migrasomes in Tspan9 flox/flox ; LysM-Cre WT/WT ( T9 f/f ; Cre W/W ) and Tspan9 flox/flox ; LysM-Cre T/T ( T9 f/f ; Cre T/T ) mouse blood by imaging flow cytometry analysis. n = 13 ( T9 f/f ; Cre W/W ) and 16 ( T9 f/f ; Cre T/T ) mice. n , Western blot analysis of nsNeuMigs isolated from T9 f/f ;Cre W/W and T9 f/f ; Cre T/T mice. Samples from ten mice were pooled and analysed together. o , Intravital imaging of neutrophil-derived migrasomes in the livers of T9 f/f ; Cre W/W and T9 f/f ; Cre T/T mice. Scale bars, 20 μm. p , Quantification of neutrophil-derived migrasomes in T9 f/f ; Cre W/W and T9 f/f ; Cre T/T mice. n = 395 ( T9 f/f ; Cre W/W ) and 410 ( T9 f/f ; Cre T/T ) cells from six mice each. q , Tail tip bleeding assay in each group. Scale bar, 1 cm. r , Statistical analysis of blood volumes. n = 18 ( T9 f/f ; Cre W/W ) and 17 (other groups) mice. All statistical data are presented as means ± s.e.m, P values were calculated using a two-tailed, unpaired t -test. The data in b , e , h , j , l , p and r were pooled from three independent experiments. The grey values of the western blots were quantified using ImageJ. Source numerical data and unprocessed blots are available in Source Data Fig. .

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Imaging, Derivative Assay, In Vitro, Western Blot, Control, Injection, Flow Cytometry, Isolation, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a-b , Flow cytometry analysis of whole blood cells from control mice and neutrophil-depleted mice ( a ) or platelet-depleted mice ( b ) after staining with FITC-anti-Ly6B.2 and PE-anti-Ly6G ( a ), or FITC-anti-CD61 and APC-anti-CD41 ( b ). Samples from five mice were pooled and analyzed together. c-d , Quantification of the number of neutrophil-derived migrasomes in ctrl and neutrophil-depleted mice revealed by imaging-flow cytometry analysis. Mouse blood samples were collected and stained with AF488-anti-Ly6G, PE-anti-CD41 and AF647-anti-Ly6B.2 for analysis. n = 5 mice for each group. e , Crude migrasomes were isolated from the same volume of blood from control or neutrophil-depleted mice, and then analyzed by western blotting using antibodies against integrin α5 and Ly6G. f , Dragonfly confocal images of crude migrasomes stained with PE-anti-Ly6G. Scale bar, 20 μm. g , Quantification of the number of neutrophil-derived migrasomes in images of crude migrasomes stained with PE-anti-Ly6G. Samples were pooled from 5 mice for each group. n = 10 field-of-views for each group. h , Dragonfly confocal images of crude migrasomes stained with Alexa Fluor 647-anti-Ly6B.2. Scale bar, 20 μm. i , Quantification of the number of neutrophil-derived migrasomes in images of crude migrasomes stained with AF647-anti-Ly6B.2. Samples were pooled from 5 mice for each group. n = 10 field-of-views for each group. j , Stitch Dragonfly confocal imaging of liver wounds in control mice, neutrophil-depleted (anti-Ly6G) mice, and neutrophil-depleted mice rescued with nsNeuMigs (i.v. 2 × 10 6 per mouse). AF488-WGA labels vessels; APC-anti-CD41 labels platelets. Scale bar, 200 μm. Dashed white lines indicate the wound boundaries. k , Statistical analysis of the relative fluorescence intensity of CD41 (platelets) enriched around the wound boundaries; n = 8 mice for each group. All of the statistical data are presented as the mean ± s.e.m, P values were calculated using the two-tailed, unpaired t-test. Gray values of western blots were quantified by Image J. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Flow Cytometry, Control, Staining, Derivative Assay, Imaging, Isolation, Western Blot, Fluorescence, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , Quantification of neutrophil-derived migrasomes in blood from WT and Tspan9 -/- mice by imaging-flow cytometry analysis; n = 12 mice for WT, n = 11 mice for Tspan9 -/- . b , Western blot analysis of nsNeuMigs isolated from the same volume blood of WT and Tspan9 -/- mice using antibodies against the indicated molecules. Samples from 10 mice were pooled and analyzed together. Gray values of western blots were quantified by Image J. c , Tail-tip bleeding assay in WT and Tspan9 -/- mice. Blood was dripped onto a clear plastic sheet for imaging. d , Statistical analysis of blood volumes. Data were pooled from four independent experiments. n = 45 mice for each group. e , Tail-tip bleeding assay in WT, Tspan9 -/- and nsNeuMig-injected (i.v. 2 × 10 6 per mouse) mice. Blood was dripped onto a clear plastic sheet for imaging. f , Statistical analysis of bleeding volume. Data were pooled from four independent experiments. n = 20 mice for each group. g , Stitch imaging of liver wounds in WT, Tspan9 -/- and nsNeuMig-injected (i.v. 2 × 10 6 per mouse) mice. AF488-WGA labels vessels; PE-anti-Ly6G (Gr1) labels neutrophils and migrasomes; APC-anti-CD41 labels platelets. Scale bar, 200 μm. Dashed white lines indicate the wound boundaries. h , Statistical analysis of relative fluorescence intensity of CD41 (platelets) enriched around the wound boundaries. n = 6 mice for the WT group; n = 7 mice for the Tspan9 -/- and Tspan9 -/- + nsNeuMig groups. i , Tail-tip bleeding assay in T9 f/f ; Cre W/W and T9 f/f ; Cre T/T mice. Blood was dripped on a clear plastic sheet for imaging. j , Statistical analysis of bleeding volumes. Data were pooled from four independent experiments. n = 43 mice in T9 f/f ; Cre W/W group; n = 42 mice in T9 f/f ; Cre T/T group. All statistical data are presented as the mean ± s.e.m. P values were calculated using the two-tailed, unpaired t-test. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Derivative Assay, Imaging, Flow Cytometry, Western Blot, Isolation, Injection, Fluorescence, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Neutrophil-derived migrasomes are an essential part of the coagulation system

doi: 10.1038/s41556-024-01440-9

Figure Lengend Snippet: a , b , Intravital imaging of neutrophils and neutrophil-derived migrasomes in the livers of control mice or mice infected with E. coli (i.p. injection of 1 × 10 9 c.f.u. in a ) or treated with LPS (i.p. injection of 10 mg kg −1 LPS in b ) for 3–4 h. Neutrophils and neutrophil-derived migrasomes were labelled with PE–anti-mouse Ly6G (Gr1; green) and blood vessels were labelled with AF647–WGA (purple). Scale bars, 20 μm. c , d , Quantification of blood neutrophil-derived migrasomes in control, LPS-treated (i.p.; 10 mg kg −1 ) and E. coli- infected (i.p.; 1 × 10 9 c.f.u.) mice using Amnis imaging flow cytometry analysis. Blood was collected 4 h after LPS or E. coli injection from the orbital sinus and stained with PE–anti-CD41 and either AF488–anti-Ly6G ( c ) or AF647–anti-Ly6B.2 ( d ) for analysis. n = 10 mice per group. The data are presented as means ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. e , Neutrophil-derived migrasomes were negatively isolated from the same volume of blood from control, LPS-treated (i.p.; 10 mg kg −1 ) or E. coli -infected (i.p;. 1 × 10 9 c.f.u.) mice and analysed by western blotting using antibodies against marker proteins for migrasomes. Ten mice per group were studied. Samples were pooled per group for subsequent analysis. The grey values of the western blots were quantified using ImageJ. f , Diagram showing how neutrophil-derived migrasomes are involved in the coagulation system. Source numerical data and unprocessed blots are available in Source Data Fig. . exp, exposure.

Article Snippet: To deplete neutrophils, mice were injected intraperitoneally with an initial 200 μg followed by 100 μg three times weekly of InVivoPlus anti-Ly6G antibody (BP0075-1; BioXCell).

Techniques: Imaging, Derivative Assay, Control, Infection, Injection, Flow Cytometry, Staining, Two Tailed Test, Isolation, Western Blot, Marker, Coagulation

Journal: PLoS Pathogens

Article Title: Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection

doi: 10.1371/journal.ppat.1007519

Figure Lengend Snippet: (A) C57BL/6, Gsdmd -/- and Casp11 -/- mice were infected intraperitoneally with 1 x 10 6 CFU of B . abortus . Mice were sacrificed at 72h, 1 and 2 weeks postinfection, and diluted spleen homogenates were added to BB medium agar plates for CFU determination. (B-D) Spleen cells from infected C57BL/6, Gsdmd -/- and Casp11 -/- mice were stained ex-vivo for flow cytometry analysis. Cells were assessed for CD11b + CD11c + (B), CD11b + F4/80 + (C) and CD11b + Ly6G + (D). Data are mean ± SD of five mice/group. (E) Splenic homogenates from mice C57BL/6, Casp11 -/- and Gsdmd -/- infected with B . abortus were submitted to a myeloperoxidase (MPO) activity assay. Data are mean ± SD of five mice/group. (F) C57BL/6, Casp11 -/- and Gsdmd -/- mice were infected with B . abortus , and 3 days post-infection were inoculated i.v. with Ly-6G PE antibody. Representative images show whole organ ex-vivo confocal images from spleens from each group. Percentage of red fluorescent pixels per organ area is also shown. Scale bar = 100 μm. (G) Analysis of CD62L MFI (median of fluorescence intensity) in C57BL/6, Casp11 -/- and Gsdmd -/- Ly6G + cell population, when stimulated with B . abortus or medium alone (NI). (H) Number of Ly6G + cells expressing IL-17 in 1x10 6 splenocytes of C57BL/6, Casp11 -/- and Gsdmd -/- mice infected two weeks with B . abortus (MOI:100). (I) Analysis of Brucella CFU in neutrophils depleted mice. Prior to and during infection, mice were treated with isotype control or with anti-Ly6G antibody. Spleens were excised at day 7 postinfection and bacterial load was measured. * p <0.05, compared to wild-type mice. The graphs are representative of two independent experiments. DCs: dendritic cells; ns: statistically not significant; NI: non-infected.

Article Snippet: Neutrophils were depleted by intraperitoneal injection of 100 μg of anti-mouse Ly6G (clone 1A8, BioXcell, West Lebanon, NH, USA) 24 hours before infection i.p. with 1 × 10 6 CFU B . abortus virulent strain S2308.

Techniques: Infection, Staining, Ex Vivo, Flow Cytometry, Activity Assay, Fluorescence, Expressing, Control