Journal: JVS-Vascular Science

Article Title: Biological mechanisms of infection resistance in tissue engineered blood vessels compared to synthetic expanded polytetrafluoroethylene grafts

doi: 10.1016/j.jvssci.2023.100120

Figure Lengend Snippet: Representative hematoxylin and eosin ( H&E ), Gram stain, and immunofluorescence images of explanted human acellular vessels ( HAV ) and expanded polytetrafluoroethylene ( ePTFE ) dialysis access conduits, determined to be infected approximately 1 year after implantation in a phase III clinical trial. H&E images show multiple cannulation tracts (blue arrows) from dialysis access through the wall of both the HAV (A-C) and ePTFE graft (G-I) . A few clusters of gram-positive bacteria (dark blue) were identified in the thrombus and fibrous tissue but not seen within the wall of the HAV explant ( D-F , green arrows). Numerous clusters of bacteria were found inside the wall (K) and within the luminal pannus tissue (L) of the ePTFE graft. The black and red dashed line boxes on the low-magnification images (A, D, G J) correspond with regions shown in color labeled high-magnification H&E and gram stain images. Immunostaining for neutrophils (green) and Staphylococcus aureus bacteria (red) in the HAV (M) and ePTFE (N) explants are shown at high magnification near regions identified with green dashed-line boxes in low-magnification images (A, G) . Neutrophils were typically found both outside and inside of the HAV (M) , but only along the exterior and not within the contaminated ePTFE graft (N) . 4′,6-Diamidino-2-phenylindole (DAPI) stained cell nuclei (blue) as well as diffuse extracellular nuclear material into edge of ePTFE (N) which suggested a loss of neutrophil viability or NETosis at this interface.

Article Snippet: Samples were stained with anti- Staphylococcus aureus ( S.aureus ) (NOVUS NB100-64,499, diluted 1:100) and anti-neutrophil elastase (R&D Systems MAB91671-100, diluted 1:50) overnight at 4°C.

Techniques: Staining, Immunofluorescence, Infection, Bacteria, Labeling, Immunostaining

Journal: JVS-Vascular Science

Article Title: Biological mechanisms of infection resistance in tissue engineered blood vessels compared to synthetic expanded polytetrafluoroethylene grafts

doi: 10.1016/j.jvssci.2023.100120

Figure Lengend Snippet: Representative Gram stain images and immunohistochemical ( IHC ) localization of neutrophils and Staphylococcus aureus ( S. aureus ) from a human acellular vessel ( HAV ) and expanded polytetrafluoroethylene ( ePTFE ) explant 2 weeks after implantation in a rodent infection model. Gram-positive S . aureus (dark blue clusters) were identified within interstices of ePTFE (B) but not seen within or around HAV explant (A) . Immunostaining for neutrophil elastase (green) and S . aureus (red) is shown in (C-F) and (G-J) . The HAV explant had only a few intact neutrophils on the tissue and graft interface (D) , but host cells (blue nuclei) infiltrated into the entire HAV wall (C and E) , unlike that of the ePTFE wall (G) . Numerous clusters of S . aureus were found within the ePTFE wall (G and I) , but no bacteria were detected in the HAV (C) . Some positive neutrophil elastase staining was observed just at the edge of the ePTFE wall, but relatively few intact nuclei were found to be associated with this staining (H) . The tissue surrounding the actively contaminated ePTFE explant (J) had a high density of neutrophil elastase-positive neutrophils compared with that of the HAV implant (F) . The green arrow in (C) shows autofluorescent red blood cells.

Article Snippet: Samples were stained with anti- Staphylococcus aureus ( S.aureus ) (NOVUS NB100-64,499, diluted 1:100) and anti-neutrophil elastase (R&D Systems MAB91671-100, diluted 1:50) overnight at 4°C.

Techniques: Staining, Immunohistochemical staining, Infection, Immunostaining, Bacteria

Journal: Parasites & Vectors

Article Title: Trichinella spiralis co-infection exacerbates Plasmodium berghei malaria-induced hepatopathy

doi: 10.1186/s13071-020-04309-6

Figure Lengend Snippet: Co-infection with T. spiralis stimulated the infiltration of CD68 + macrophages and elastase-expressed neutrophils in the liver and spleen of mice infected with Pb ANKA. Immunohistochemical staining for CD68 + macrophages ( a ) and neutrophil elastase-positive cells ( b ), and semi-quantitative analysis of the expression levels of CD68 + macrophages ( c ) and neutrophil elastase ( d ) in the liver and spleen of different groups of mice on day 22 after T. spiralis infection and/or on day 13 after Pb ANKA infection. Shown are immunohistochemical staining for CD68 + macrophages or elastase-expressed neutrophils in the liver and spleen of an uninfected mouse, a T. spiralis -mono-infected mouse, a Pb ANKA-mono-infected mouse, and a T. spiralis + Pb ANKA-co-infected mouse. The original magnification was 400× ( Scale-bar : 50 µm). Data are presented as means ± SD ( n = 6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 vs naive mice; ## P < 0.01 and ### P < 0.001 vs Pb ANKA-mono-infected mice

Article Snippet: After washing with PBS, sections were incubated with the primary antibodies including CD68 (1:200, Bsm-33056m; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), neutrophil elastase (1:100, PB1114; Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China), galectin (Gal)-1 (1:250, A00470; Wuhan Boster Biological Engineering Co., Ltd.), or Gal-3 (1:200, Bs-0721r; Beijing Biosynthesis Biotechnology Co., Ltd.) at 37 °C for 1 h. Negative controls were performed without a primary antibody.

Techniques: Infection, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata promoted migration of neutrophils to infected wound. (A) Experimental design. (B) Flow cytometry analysis of neutrophils in the bone marrow. (C) Percentage of neutrophils in bone marrow leukocytes. (D) Flow cytometry analysis of neutrophils in peripheral blood leukocytes. (E) Percentage and (F) number of neutrophils in peripheral blood leukocytes. The data are presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Migration, Infection, Flow Cytometry, Control

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Neutrophil infiltration in the infected wound skin of mice. (A) H&E was used to observe neutrophil infiltration in the infected wound skin. (B) Immunohistochemistry of MPO expression was used to observe the number of neutrophils in the infected wound skin at 6 h post-infection. (C) The number of MPO-expressing positive cells in each group of mice was counted based on the location and area of the frame-selected skin-infected wounds in this figure (B) . (D) Number of MRSA in the infected wound skin. Data were presented as the mean ± SD, n = 3. # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Infection, Immunohistochemistry, Expressing

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata promoted migration of neutrophils to air pouch. (A) Experimental design. (B, C) Percentage and (D) number of neutrophils in peripheral blood leukocytes and flow cytometric analysis of CD45 + cells in peripheral blood leukocytes are shown in . (E) Flow cytometric analysis of neutrophils in air pouch leukocytes. (F) Percentage and (G) number of neutrophils in air pouch leukocytes. (H) Number of MRSA in the air pouch. Data were presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Migration, Control

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata promoted activation of neutrophils in the infected air pouch. (A) Experimental design. (B) Flow cytometric analysis of the ROS level in neutrophils. The mean fluorescence intensity of DCF was used to evaluate ROS levels in neutrophils. (C) Mean fluorescence intensity of DCF in neutrophils. (D) NET area in this figure (E, F) . NET formation in the air pouch in (E) Model and (F) Lanata groups was observed by immunofluorescence staining. Scale bar: 100 μm. Data were presented as the mean ± SD, n = 3. # p < 0.05 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Activation Assay, Infection, Fluorescence, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata regulated mRNA expression in neutrophils. (A) Experimental design. (B) Pie chart of differentially expressed genes between the Lanata and MRSA groups. (C) Cluster heat map, (D) GO, and (E) KEGG enrichment analysis of DEGs between the Lanata and MRSA groups.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata upregulated mRNA and protein expressions of CXCR2, PSGL-1, and MPO in neutrophils in peripheral blood. (A) Experimental design. (B–D) The mRNA expression levels of CXCR2, PSGL-1, and MPO in neutrophils were detected by RT-qPCR. (E) The protein expression of CXCR2 and MPO in neutrophils was detected by flow cytometry. Median fluorescence intensity of (F) CXCR2 and (G) PSGL-1 in neutrophils. (H) The concentration of MPO in neutrophils was detected by ELISA. Data were presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Lanata upregulated mRNA and protein expressions of CXCR2, PSGL-1, and MPO in neutrophils in the air pouch. (A–C) The mRNA expressions of CXCR2, PSGL-1, and MPO in neutrophils were detected by RT-qPCR. (D) The protein expression of CXCR2 and MPO in neutrophils was detected by flow cytometry. The median fluorescence intensity of (E) CXCR2 and (F) PSGL-1 in neutrophils. (G) The concentration of MPO in neutrophils was detected by ELISA. Data were presented as the mean ± SD, n = 3. # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Panzerina lanata accelerates methicillin-resistant Staphylococcus aureus eradication by promoting migration and activation of neutrophils

doi: 10.3389/fphar.2024.1501744

Figure Lengend Snippet: Inhibition of CXCR2 and MPO significantly attenuated the effect of Lanata on promoting migration and activation of neutrophils. (A) Experimental design. (B) Immunofluorescence staining was used to observe the NET formation in the air pouch of mice. Scale bar: 100 μm. (C, D) Mean fluorescence intensity of DCF in neutrophils in the air pouch of mice was detected by flow cytometry at 6 h post-infection. (E, F) The number of neutrophils in the air pouch was detected by flow cytometry at 6 h post-infection. Data were presented as the mean ± SD, n = 4. # p < 0.05, ## p < 0.01 vs. MRSA group.

Article Snippet: The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

Techniques: Inhibition, Migration, Activation Assay, Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Infection