Journal: bioRxiv
Article Title: Detrimental Influence of Arginase-1 in Infiltrating Macrophages on Post-Stroke Functional Recovery and Inflammatory Milieu
doi: 10.1101/2024.07.08.602449
Figure Lengend Snippet: (A) A schematic diagram of the experimental design. A chunk of the cortical tissue, including the infarcted and spared peri-infarct cortex, was obtained, and the infarct core was carefully dissected and removed. The remaining peri-infarct cortical tissue was dissociated into single cells using the Adult brain dissociation kit, and the dissociated cells were subjected to FACS to isolate CD11b+/CD45 low microglial cells from macrophages. (B) A heatmap plot comparing all detected cytokine expressions between flox and cKO groups in the sham or stroke conditions. Log 2 (fold change) of the gene expression value of each cKO subject compared to the average expression value of the flox group was color-coded. Asterisks indicate the statistical significance of comparing cKO and flox groups in the stroke condition normalized by subtracting the difference between cKO and flox groups in the sham conditions. * and ** indicate p < 0.05 and p < 0.01, respectively, by unpaired T-test. (C) A volcano plot of DEGs by Arg1 cKO in the stroke condition. Significantly regulated genes were determined based on log 2 (fold change) > 0.5 and p < 0.05 criteria. Down DEGs were shown in blue, and up DEGs in red. The fold change value comparing cKO and flox control animals in the stroke condition was normalized by subtracting the difference between cKO and flox groups in the sham condition. (D) K-means clustering of the significant cytokine DEGs. (E) The degree of interactions within each cluster was calculated using the web-based STRING analysis. Protein interaction value was obtained by dividing the number of node degrees by the total number of nodes. (F) A bubble plot of the enriched GO processes ( p < 0.05 = FDR < 95.0). The bubble size indicates the number of annotated genes in the GO process. (G) Representative images of semi-PCR products of microglial phagocytic markers (CD68, CR3, CD36, GAL-3, and TREM2). mRNA samples were obtained from FACS-isolated microglia out of the peri-infarct cortex 7 days post-stroke. (H-L) Quantitative comparison of mRNA expressions by real-time PCR. CD68 and CR3 showed significantly reduced expression in Arg1 cKO animals compared to flox animals after stroke. *, ** and*** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, by One-way ANOVA followed by post hoc Tukey’s multiple comparison.
Article Snippet: For FACS antibody labeling, anti-CD11b-Alexa488 (Bio-rad, BRMCA275A488) and anti-CD45-Alexa647 (Bio-rad, BRMCA43A647) antibodies were incubated (10 μl per 1.0 x 10 6 cells) for 30 minutes in an ice bucket covered with aluminum foil to protect the samples from light exposure.
Techniques: Expressing, Control, Isolation, Comparison, Real-time Polymerase Chain Reaction
Bio-Rad is a verified supplier 
