Journal: Neural Regeneration Research

Article Title: Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

doi: 10.4103/1673-5374.392889

Figure Lengend Snippet: Microglia are a crucial source of hypochlorous acid in the stroke recovery phase. (A–C) Co-localization and statistical analysis of Iba-1 + microglia (Cy3, red) and hypochlorous acid (HKOCl-3, green) in ischemic SVZ of MCAO rats. Hypochlorous acid was visualized using a HKOCl-3 probe. Hypochlorous acid levels (normalized to day 1) increased on the 3 rd and 5 th days after MCAO. Fluorescence intensity of Iba-1 increased on the 5 th day, but decreased on the 7 th and 14 th days after MCAO. Scale bar: 100 μm. (D) Hypochlorous acid production in BV2 cells and neutrophils with or without O/R challenge. (E) BV2/C17.2 co-culture system to examine the effect of hypochlorous acid produced by BV2 cells, with or without O/R challenge. (F) Neutrophil/C17.2 co-culture system to examine the effect of hypochlorous acid produced by neutrophils on C17.2 cell viability under O/R conditions. Taurine (100 μM) and 4-ABAH (100 μM) were used to scavenge or prevent hypochlorous acid production, respectively, in O/R-challenged neutrophils. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (B, C: one-way analysis of variance followed by Dunnett’s post hoc test; D–F: one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4,6-Diamidino-2-phenylindole; Iba1: ionized calcium binding adapter molecule 1; O/R: oxygen-glucose deprivation/reoxygenation; SVZ: subventricular zone.

Article Snippet: The mouse NSC line C17.2 (kindly provided by Professor Jiangang Shen, RRID: CVCL_4511), microglia cell line BV2 (Cat# 305156, CLS Cell Lines Service GmbH, Eppelheim, Germany, RRID: CVCL_0182), and hippocampal neuronal cell line HT22 (Cat# 305158, CLS Cell Lines Service GmbH, RRID: CVCL_0321) were cultured in complete high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin.

Techniques: Fluorescence, Co-Culture Assay, Produced, Binding Assay

Journal: Neural Regeneration Research

Article Title: Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

doi: 10.4103/1673-5374.371368

Figure Lengend Snippet: Increased Vav1 expression in BV-2 cells subjected to OGD/R. (A) Vav1 mRNA expression in control BV-2 cells and BV-2 cells subjected to OGD/R. (B) Vav1 protein expression in control BV-2 cells and BV-2 cells subjected to OGD/R. (C) Immunofluorescence staining for Vav1 in control BV-2 cells and BV-2 cells subjected to OGD/R. Compared with the control cells, Vav1 expression was increased in OGD/R cells. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (D) Quantification of Vav1-positive cells. (E) Vav1 mRNA expression in Vav1 knockdown BV-2 cells. (F) Vav1 protein expression in Vav1 knockdown BV-2 cells. Data are expressed as the mean ± SD, and were analyzed by unpaired t -test (A, B, D) or one-way analysis of variance followed by Tukey’s multiple comparisons test (E, F). The experiments were repeated three times. DAPI: 4′,6-Diamidino-2-phenylindole; OGD/R: oxygen-glucose deprivation/reoxygenation; Vav1: Vav guanine nucleotide exchange factor 1.

Article Snippet: The mouse microglia cell line BV-2 (iCELL, Shanghai, China, Cat# iCell-m011, RRID: CVCL_0182) was used as an in vitro subject to further confirm the effect of Vav1 on microglial and NLRP3 inflammasome activation.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Neural Regeneration Research

Article Title: Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

doi: 10.4103/1673-5374.371368

Figure Lengend Snippet: Vav1 knockdown represses inflammation in BV-2 cells subjected to OGD/R. (A) IL-1 β, TNF-α , and IL-18 mRNA levels in control BV-2 cells and BV-2 cells subjected to OGD/R were analyzed by qPCR. (B) The contents of IL-1β, TNF-α, and IL-18 in control BV-2 cells and BV-2 cells subjected to OGD/R were detected by ELISA. (C) Immunofluorescence staining for NLRP3 (FITC, green) in BV-2 cells subjected to OGD/R. Compared with control cells, NLRP3 expression was increased in BV-2 cells subjected to OGD/R, while inhibition of Vav1 expression decreased NLRP3 expression. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (D) Quantification of NLRP3 positive cells. (E, F) NLRP3, ASC, and cleaved caspase-1 (p20) protein levels in BV-2 cells subjected to OGD/R were analyzed by western blot. Data are expressed as the mean ± SD, and were analyzed by one-way analysis of variance followed by Tukey’s multiple comparisons test. The experiments were repeated three times. ASC: Apoptosis-associated speck-like protein; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothiocyanate; IL-18: interleukin-18; IL-1β: interleukin-1β; NLRP3: NOD-like receptor pyrin 3; OGD/R: oxygen-glucose deprivation/reoxygenation; qPCR: quantitative polymerase chain reaction; TNF-α: tumor necrosis factor-α; Vav1: Vav guanine nucleotide exchange factor 1.

Article Snippet: The mouse microglia cell line BV-2 (iCELL, Shanghai, China, Cat# iCell-m011, RRID: CVCL_0182) was used as an in vitro subject to further confirm the effect of Vav1 on microglial and NLRP3 inflammasome activation.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing, Inhibition, Western Blot, Real-time Polymerase Chain Reaction

Journal: EMBO Reports

Article Title: Ganglioside GT1b prevents selective spinal synapse removal following peripheral nerve injury

doi: 10.1038/s44319-025-00452-2

Figure Lengend Snippet: ( A ) Experimental timeline for phagocytosis assays using BV2 and primary mixed glial cells after GT1b stimulation. ( B ) Overview of live cell imaging and flow cytometry for the phagocytosis assay. ( C ) Representative image of the glial phagocytosis of E. coli -conjugated pHrodo bioparticles after GT1b treatment. Scale bar = 500 µm. ( D ) Quantification of glial phagocytic activity. P value (30 min); Control vs. GT1b[100 µg/ml] = 0.0136. P value (40 min); Control vs. GT1b[100 µg/ml] = 0.0017. P value (50 min); Control vs. GT1b[100 µg/ml] = 0.0003. P value (60 min); Control vs. GT1b[10 µg/ml] = 0.0333, Control vs. GT1b[100 µg/ml] <0.0001. P value (70 min); Control vs. GT1b[10 µg/ml] = 0.02, Control vs. GT1b[100 µg/ml] <0.0001. P value (80 min); Control vs. GT1b[1 µg/ml] = 0.0414, Control vs. GT1b[10 µg/ml] =0.0117, Control vs. GT1b[100 µg/ml] <0.0001. n ; Control =3, GT1b[1 µg/ml] = 4, GT1b[10 µg/ml] = 4, GT1b[100 µg/ml] = 4. ( E ) Gating strategy for BV2 cells and ( G ) mixed glia, with corresponding phagocytic activity levels depicted in ( F ) and ( H ), respectively. P value ( F : M.F.I); CTL vs. 1 µg/ml = 0.2108, CTL vs. 10 µg/ml <0.0001, CTL vs. 100 µg/ml <0.0001. P value ( F : cell count); CTL vs. 1 µg/ml = 0.9947, CTL vs. 10 µg/ml = 0.0041, CTL vs. 100 µg/ml <0.0001. n ( F ); CTL = 3, 1 µg/ml = 3, 10 µg/ml = 3. 100 µg/ml = 3. P value ( H : microglia_M.F.I); CTL vs. 1 µg/ml = 0.0342, CTL vs. 10 µg/ml = 0.0341, CTL vs. 100 µg/ml <0.0001. P value ( H : microglia_cell count); CTL vs. 1 µg/ml = 0.1046, CTL vs. 10 µg/ml = 0.3841, CTL vs. 100 µg/ml = 0.0349. P value ( H : astrocyte_M.F.I); CTL vs. 1 µg/ml = 0.0198, CTL vs. 10 µg/ml = 0.0611, CTL vs. 100 µg/ml =0.0015. P value ( H : astrocyte_cell count); CTL vs. 1 µg/ml = 0.003, CTL vs. 10 µg/ml = 0.0598, CTL vs. 100 µg/ml <0.0001. n ( H ); CTL = 3, 1 µg/ml = 4, 10 µg/ml = 4. 100 µg/ml = 4. ( I ) Western blot representation and ( J ) quantification of phosphorylated and total SYK levels in primary mixed glia. P value; Control vs. 10 µg/ml = 0.0432, CTL vs. 100 µg/ml = 0.07. n ; Cont = 4, 10 µg/ml = 4. 100 µg/ml = 4. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test and two-way ANOVA with Dunnett’s multiple comparison test. .

Article Snippet: BV2 cell line , Accegen , Cat # ABV-TC212S.

Techniques: Live Cell Imaging, Flow Cytometry, Phagocytosis Assay, Activity Assay, Control, Cell Counting, Western Blot, Comparison