bv2 mouse microglia cell line  (ImmunoTools)

Journal: Neural Regeneration Research

Article Title: Inhibiting SHP2 reduces glycolysis, promotes microglial M1 polarization, and alleviates secondary inflammation following spinal cord injury in a mouse model

doi: 10.4103/NRR.NRR-D-23-01925

Figure Lengend Snippet: SHP2 downregulation reduces microglia-mediated inflammation. (A, B) mRNA expression levels of SHP2, iNOS, CD86, TNF-α, IL-1β, CD206, and Arg-1 in microglia treated with NSC87877 and siSHP2, as assessed by quantitative polymerase chain reaction. (C–E) Immunoreactivities of CD163 (green-CoraLite488) and CD86 (red-CoraLite594) in microglia treated with NSC87877, as detected by immunofluorescence staining. Microglia treated with LPS showed a significant increase in expression of the inflammatory marker CD86, while BV2 cells treated with LPS + NSC87877 exhibited a significant decrease in CD86 expression. Additionally, there was a significant increase in expression of the anti-inflammatory marker CD163 in the LPS + NSC87877 group relative to the LPS group. Scale bars: 50 μm. (F–I) Western blot for the protein expressions of SHP2, iNOS, CD86, CD206, Arg-1, and other proteins in microglia treated with NSC87877 and siSHP2. Data are expressed as the mean ± SD. The experiment was repeated three times. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni’s post hoc test). Arg-1: Arginase-1; CD163: cluster of differentiation 163; CD206: cluster of differentiation 206; CD86: cluster of differentiation 86; DAPI: 4′,6-diamidino-2′-phenylindole; IF: immunofluorescence staining; IL-1β: interleukin-1 beta; LPS: lipopolysaccharide; NS: not significant; NSC87877: SHP2 inhibitor; TNF-α: tumor necrosis factor-alpha; SHP2: Src homology 2 domain-containing protein tyrosine phosphatase 2; siNC: small interfering RNA negative control; siSHP2: small interfering RNA of SHP2.

Article Snippet: The PC12 neuronal cell line (Cat# CL-0481, RRID:CVCL_0481), and the BV2 mouse microglia cell line (Cat# CL0493, RRID: CVCL_0182) were purchased from Procell Life Science and Technology Co. (Wuhan, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Marker, Western Blot, Small Interfering RNA, Negative Control

Journal: Neural Regeneration Research

Article Title: Inhibiting SHP2 reduces glycolysis, promotes microglial M1 polarization, and alleviates secondary inflammation following spinal cord injury in a mouse model

doi: 10.4103/NRR.NRR-D-23-01925

Figure Lengend Snippet: SHP2 inhibition reduces neuronal apoptosis in the context of SCI. (A, B) NeuN (red-CoraLite594) and SHP2 (green-CoraLite488) immunoreactivities in mice treated with NSC87877 14 days after SCI, as detected by immunofluorescence. Quantitative analysis showing that a decrease in SHP2 fluorescence intensity and an increase in the fluorescence intensity of NeuN, a neuronal marker, in SCI + NSC87877 group mice near the injury site ( n = 5 slices from three mice per group). Scale bars: 50 μm. (C) Nissl staining of mouse spinal cord transverse and longitudinal sections 14 days after SCI showing that, compared with the SCI group, the SCI + NSC87877 group exhibited more Nissl bodies with a more uniform distribution ( n = 5 slices from three mice per group). Scale bars: 100 μm (left), 600 μm (right). (D, E) Western blot analysis of Caspase3, C-caspase3, Bax, and Bcl-2 protein expression in PC12 cells co-cultured with BV2 cells treated with LPS or LPS + NSC87877 for 24 hours. The experiment was repeated three times. (F, G) C-caspase3 (red-CoraLite594) expression in PC12 cells was detected by immunofluorescence staining. Quantitative analysis showed decreased immunofluorescence intensity of the apoptosis marker C-caspase3 in neurons co-cultured with BV2 cells from the LPS + NSC87877 group. The experiment was repeated three times. Scale bars: 50 μm. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Bonferroni’s post hoc test). Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; Caspase3: cysteine-aspartic acid protease 3; C-caspase3: cleaved caspase-3; DAPI: 4′,6-diamidino-2′-phenylindole; IF: immunofluorescence staining; LPS: lipopolysaccharide; NeuN: neuronal nuclei; NSC87877: SHP2 inhibitor; SCI: spinal cord injury; SHP2: Src homology 2 domain-containing protein tyrosine phosphatase 2.

Article Snippet: The PC12 neuronal cell line (Cat# CL-0481, RRID:CVCL_0481), and the BV2 mouse microglia cell line (Cat# CL0493, RRID: CVCL_0182) were purchased from Procell Life Science and Technology Co. (Wuhan, China).

Techniques: Inhibition, Immunofluorescence, Fluorescence, Marker, Staining, Western Blot, Expressing, Cell Culture