neutrophil magnetic bead negative selection kit  (STEMCELL Technologies Inc)

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: Intramuscular ectopic endochondral ossification induced by fracture hematoma and IL‐8 were similar at the early stage, recruiting neutrophils first and BMSCs and macrophages at a later stage. A) Histological observation of intramuscular implantation of 2‐day and 4‐day cranial defect hematoma using Masson's trichrome staining. 4‐day hematoma‐induced ectopic endochondral ossification. B) b1) In vitro release profile of IL‐8 from gelatin sponge. IL‐8 loaded onto gelatin sponge was rapidly released within 24 h. b2) Time‐course cytometric quantification of myeloid cell subpopulations in the implant using flow cytometry. Neutrophils promptly appeared and peaked at day 1, while BMSCs and macrophages peaked at day 5. Histological observation at day 7 using b3) Masson's trichrome staining and b4) Safranin‐O staining. Ectopic cartilage was formed around the implant. b5) Type II collagen (Col II), type I collagen (Col I), and alkaline phosphatase (ALP) immunohistochemistry staining. The exclusive cartilaginous Col II, together with the osteogenic‐related Col I and ALP indicated ectopic endochondral ossification. ( n = 3 for each group)

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: Staining, In Vitro, Flow Cytometry, Immunohistochemistry

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: Neutrophils were pivotal for bone‐healing at the initiating stage. α ‐Ly6G or α ‐F4/80 antibody was continuously injected from 2 days pre‐ to 7 days post‐cranial defect creation for depletion of neutrophils or macrophages (referred to as N‐ and M‐), respectively, with IgG antibody as a control. A) Micro‐CT reconstruction of the defect site after 90 days. Quantification histogram of B) bone volume/total volume (BV/TV), C) bone mineral density (BMD), D) trabecular thickness (Tb.Th.), E) trabecular space (Tb.Sp.), and F) trabecular number (Tb.N.). (** p < 0.01, * p < 0.05; n = 3 for each group)

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: Injection, Control, Micro-CT

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: Depletion of circulatory neutrophils cut off the IL‐8‐induced myeloid cell recruitment and ectopic endochondral ossification. A) Flow cytometry of the ectopic implant and its quantitative histogram of B) neutrophils, C) macrophages, and D) BMSCs. Depletion of neutrophils inhibited recruitment of all myeloid cells. E) Numbers of neutrophils and BMSCs exhibited a positive correlation. F) Implants retrieved at day 7, G) histological observation using Masson's trichrome staining, and H) quantification of cell‐enwrapped areas. Control group induced ectopic cartilage formation, while N‐ and M‐ significantly decreased the cell‐wrapped area and prevented further differentiation. (*** p < 0.001, ** p < 0.01, * p < 0.05; n = 5 for each group)

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: Flow Cytometry, Staining, Control

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: Interaction of neutrophils and BMSCs were key to anabolism, while macrophages were essential for catabolism. Irradiated mice received IL‐8‐gelatin implantation and injection of exogenous myeloid cells. A) Quantification of cell‐enwrapped areas and B) histological observation using Masson's trichrome staining. C) Fluorescent‐labeled neutrophils and BMSCs around the implant. When neutrophils and BMSCs were simultaneously transfused (N+B), greater cell recruitment and ECM were observed around the implant; when only macrophages were transfused the implant was rapidly degraded. (Compared with ctrl: *** p < 0.001, ** p < 0.01, * p < 0.05; n = 5 for each group).

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: Irradiation, Injection, Staining, Labeling

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: IL‐8 (10 ng mL −1 ) polarized neutrophils toward the N2 phenotype, which secreted SDF‐1 α to mediate BMSC recruitment and differentiation via the SDF‐1/CXCR4 axis and its downstream PI3K/AKT pathway and β ‐catenin‐mediated migration. A) Expressions of proinflammatory and anti‐inflammatory genes in neutrophils under different concentrations of IL‐8. B) Heat map of mouse inflammatory response and autoimmunity profiler PCR array: Untreated (Ctr), IL‐8‐, and LPS‐treated neutrophils (10 ng mL −1 , 24 h exposure). Data are row‐relative and normalized to GAPDH. C) Transwell assay highlighting BMSC recruitment by IL‐8, neutrophils, and IL‐8‐treated neutrophils (migrated cells counted in five random 200 × microscopic fields). D) Mouse cytokine antibody array analyses of conditioned media from neutrophils or IL‐8‐treated neutrophils (10 ng mL −1 , 24 h exposure) and the top four upregulated cytokines quantified using densitometry. E) ELISA‐determined SDF‐1 α secreted by neutrophils treated with IL‐8 at different concentrations. F) Recruitment of BMSC pretreated with CXCR4 antagonist AMD3100 and PI3K/AKT inhibitor LY294002. G) SDF‐1‐induced phosphorylation of Akt was inhibited by AMD3100 or LY294002. H) Immunofluorescent staining of β ‐catenin translocated to nuclei by SDF‐1 α and inhibited by AMD3100 or LY294002. I) Irradiated mice received IL‐8‐gelatin implantation and injection of exogenous neutrophils and BMSCs pretreated with AMD3100 or LY294002. (Compared with ctrl: *** p < 0.001, ** p < 0.01, * p < 0.05. Compared with 10 ng mL −1 : ### p < 0.001, ## p < 0.01, # p < 0.05; n = 3 for each group in PCR and ELISA; n = 5 for each group in transwell assay).

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: Migration, Transwell Assay, Ab Array, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Staining, Irradiation, Injection

Journal: Advanced Science

Article Title: N2‐Polarized Neutrophils Guide Bone Mesenchymal Stem Cell Recruitment and Initiate Bone Regeneration: A Missing Piece of the Bone Regeneration Puzzle

doi: 10.1002/advs.202100584

Figure Lengend Snippet: A) The role of neutrophils in the initiation process of bone regeneration elucidated via in vivo ectopic simulation of endochondral ossification. The ectopic implantation of IL‐8 at a high and medium level have simulated the microenvironment of bone fracture at the early and later stage, respectively. High‐level IL‐8 at the early stage led to a proinflammatory microenvironment and phagocytosis to eliminate the damaged tissue debris for inflammation resolution. Thereafter, an adequate level of IL‐8 at the later stage polarizes neutrophils to an N2‐subtype for endochondral ossification. B) Signaling pathway of BMSC recruitment by neutrophils. N2‐polarized neutrophils release SDF‐1 α to recruit BMSCs via the SDF‐1/CXCR4 axis, its downstream PI3K/Akt pathway, and β ‐catenin‐mediated migration.

Article Snippet: A mouse neutrophil isolation negative magnetic bead selection kit (BM, Catalog. 480 058, MojoSort, BioLegend) and a mouse monocyte isolation kit (BM, Order no. 130‐100‐629, Miltenyi Biotec) were used for isolation of neutrophils and monocytes, respectively, according to the manufacturer's instructions.

Techniques: In Vivo, Migration