Journal: Aging and Disease

Article Title: Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke

doi: 10.14336/AD.2023.0505

Figure Lengend Snippet: Transcriptional landscape of microglia at the acute stage of ischemic stroke. (A) Schematic diagram of sample acquisition, single-cell RNA sequencing, and microglia identification. (Staining by Tmem119). (B) Uniform Manifold Approximation and Projection (UMAP) plot of the microglial subpopulations. (C) The temporal distribution of microglial cells in the UMAP plot. (D) Bar plot of the temporal abundance proportion of each subpopulation. (E) Dot plot displays signature markers of each subpopulation. (F) Heatmap of gene set variation analysis (GSVA) pathway enrichment for each subpopulation. (G) Heatmap of highly activated transcription factors (TF) for each microglial subpopulation.

Article Snippet: After the process of serum blocking, the antibody of the signature marker of each subpopulation was used for double staining, including Hpgd (the marker for Mic_home; Affinity; DF14850; 1:100), P2ry13 (the marker for Mic_pre1; allomone; APR-017; 1:100), Wsb1 (the marker for Mic_pre2; Affinity; DF12505; 1:100), Nfkbiz (the marker for Mic_M1L1; Affinity; DF12429; 1:100), Cd83 (the marker for Mic_M1L2; Affinity; AF5233; 1:400), Arhgap45(the marker for Mic_np1; proteintech; 14832-1-AP; 1:100), Rgs10 (the marker for Mic_np2;Affinity; DF4414; 1:100), and Pkm (the marker for Mic_np3; Affinity; DF5234; 1:100).

Techniques: RNA Sequencing, Staining

Journal: Aging and Disease

Article Title: Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke

doi: 10.14336/AD.2023.0505

Figure Lengend Snippet: Subpopulation analysis of microglia clusters under homeostatic conditions. (A) Pseudotime plot (from monocle3) shows the differentiation trajectory from Mic_home to Mic_pre2 cells. (B) Line plot of the fraction of cells in the Mic_home, Mic_pre1, and Mic_pre2 subpopulations over time. (C) Expression of the ten genes with the highest Moran’s I score alongside the pseudotime trajectory. (D) Venn plot of the mutual and unique marker genes for the Mic_home, Mic_pre1, and Mic_pre2 subpopulations. (E) Volcano plot displays the differentially expressed genes between Mic_home and Mic_pre2 cells. Red dots represent the upregulated genes in the Mic_home cluster compared with the Mic_pre2 cluster and blue dots represent the downregulated genes. (F) Violin plot of the gene set variation analysis (GSVA) scores for important functional pathways of the Mic_home, Mic_pre1, and Mic_pre2 subpopulations. (G) Double immunofluorescence staining of Mic_home cells at each sampling time. Coronal brain sections are all stained with anti-Iba1(green, representing microglia), DAPI (blue, representing cell nuclei), and Hpgd (red, representing the marker of Mic_home cells) antibodies (N=3). The yellow bar represents 100μm. (H) Double immunofluorescence staining of Mic_pre2 cells at each sampling time. Coronal brain sections are all stained with anti-Iba1(green, representing microglia), DAPI (blue, representing cell nuclei), and Wsb1 (red, representing the marker of Mic_pre2 cells) antibodies (N=3). The yellow bar represents 100μm. (I) Bar plot of the ratio of Hpgd + Iba1 + cells (green plus red) compared with all DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6). (J) Bar plot of the ratio of Wsb1 + Iba1 + cells (green plus red) compared with DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6).

Article Snippet: After the process of serum blocking, the antibody of the signature marker of each subpopulation was used for double staining, including Hpgd (the marker for Mic_home; Affinity; DF14850; 1:100), P2ry13 (the marker for Mic_pre1; allomone; APR-017; 1:100), Wsb1 (the marker for Mic_pre2; Affinity; DF12505; 1:100), Nfkbiz (the marker for Mic_M1L1; Affinity; DF12429; 1:100), Cd83 (the marker for Mic_M1L2; Affinity; AF5233; 1:400), Arhgap45(the marker for Mic_np1; proteintech; 14832-1-AP; 1:100), Rgs10 (the marker for Mic_np2;Affinity; DF4414; 1:100), and Pkm (the marker for Mic_np3; Affinity; DF5234; 1:100).

Techniques: Expressing, Marker, Functional Assay, Double Immunofluorescence Staining, Sampling, Staining

Journal: Aging and Disease

Article Title: Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke

doi: 10.14336/AD.2023.0505

Figure Lengend Snippet: The comparison between M1-polarization-like subpopulations and clusters under homeostatic state. (A) Feature plot of the representative genes for M1 and M2 polarization. (B) Line plot to compare the proportional amount of Mic_home, Mic_pres, Mic_M1L1, and Mic_M1L2 cells over time. (C) Relative fraction of cells at S and G2/M phases in Mic_pre1, Mic_pre2, Mic_M1L1, and Mic_M1L2 subpopulations. The total value of the Mic_home is regarded as 1. (D) Volcano plot displaying the differentially expressed genes between Mic_M1Ls and Mic_pres clusters. The genes are shown in different colors according to the subpopulation with the higher expression. (E) Volcano plot displaying the differentially expressed genes between Mic_pre2 and Mic_M1L2 clusters. Red dots represent the upregulated genes in the Mic_pre2 cluster compared with the Mic_M1L2 cluster and blue dots represent the downregulated. (F) Volcano plot displaying the differentially expressed genes between Mic_home and Mic_M1L1 subpopulations. Red dots represent the upregulated genes in the Mic_home cluster compared with the Mic_M1L1 cluster and blue dots represent the downregulated.

Article Snippet: After the process of serum blocking, the antibody of the signature marker of each subpopulation was used for double staining, including Hpgd (the marker for Mic_home; Affinity; DF14850; 1:100), P2ry13 (the marker for Mic_pre1; allomone; APR-017; 1:100), Wsb1 (the marker for Mic_pre2; Affinity; DF12505; 1:100), Nfkbiz (the marker for Mic_M1L1; Affinity; DF12429; 1:100), Cd83 (the marker for Mic_M1L2; Affinity; AF5233; 1:400), Arhgap45(the marker for Mic_np1; proteintech; 14832-1-AP; 1:100), Rgs10 (the marker for Mic_np2;Affinity; DF4414; 1:100), and Pkm (the marker for Mic_np3; Affinity; DF5234; 1:100).

Techniques: Comparison, Expressing

Journal: Aging and Disease

Article Title: Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke

doi: 10.14336/AD.2023.0505

Figure Lengend Snippet: Subpopulation analysis of M1-polarization-like microglia clusters. (A) Pseudotime plot (from monocle3) shows the differentiation trajectory between Mic_M1L1 and Mic_M1L2 cells. (B) Line plot of the fraction of cells in Mic_M1L1 and Mic_M1L2 subpopulations over time. (C) Expression of the ten genes with the highest Moran’s I score alongside the pseudotime trajectory. (D) Violin plot of the functional pathways revealed by gene set variation analysis (GSVA) in the Mic_M1L1 and Mic_M1L2 subpopulations. (E) Volcano plot displays the differentially expressed genes between Mic_M1L1 and Mic_M1L2 cells. Red dots represent the upregulated genes in the Mic_M1L1 cluster compared with the Mic_M1L2 cluster and blue dots represent the downregulated. (F) Relative activity of the highly expressed transcription factors (TF) in Mic_M1L2 cells compared with Mic_M1L1 cells. (G) Double immunofluorescence staining of Mic_M1L1 cells at each sampling time. Coronal brain sections are all stained with anti-Iba1(green, representing microglia), Nfkbiz (red, the marker of Mic_M1L1 cells), and DAPI (blue, representing cell nuclei) antibodies (N=3). The yellow bar represents 100μm. The arrows point to the cells simultaneously marked by Iba1 and Nfkbiz. (H) Double immunofluorescence staining of Mic_M1L2 cells at each sampling time. Coronal brain sections are all stained with anti-Iba1(green, representing microglia), Cd83 (red, the marker of Mic_M1L2 cells), and DAPI (blue, representing cell nuclei) antibodies (N=3). The yellow bar represents 100μm. The arrows point to the cells simultaneously marked by Iba1 and Cd83. (I) Bar plot of the ratio of Nfkbiz + Iba1 + cells (green plus red) compared with all DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6). (J) Bar plot of the ratio of Cd83 + Iba1 + cells (green plus red) compared with all DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6).

Article Snippet: After the process of serum blocking, the antibody of the signature marker of each subpopulation was used for double staining, including Hpgd (the marker for Mic_home; Affinity; DF14850; 1:100), P2ry13 (the marker for Mic_pre1; allomone; APR-017; 1:100), Wsb1 (the marker for Mic_pre2; Affinity; DF12505; 1:100), Nfkbiz (the marker for Mic_M1L1; Affinity; DF12429; 1:100), Cd83 (the marker for Mic_M1L2; Affinity; AF5233; 1:400), Arhgap45(the marker for Mic_np1; proteintech; 14832-1-AP; 1:100), Rgs10 (the marker for Mic_np2;Affinity; DF4414; 1:100), and Pkm (the marker for Mic_np3; Affinity; DF5234; 1:100).

Techniques: Expressing, Functional Assay, Activity Assay, Double Immunofluorescence Staining, Sampling, Staining, Marker

Journal: Aging and Disease

Article Title: Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke

doi: 10.14336/AD.2023.0505

Figure Lengend Snippet: Subpopulation analysis of neuropeptide-associated microglia clusters. (A) Pseudotime plot (from monocle3) shows the differentiation trajectory in Mic_np1, Mic_np2, and Mic_np3 cells respectively. (B) Line plot of the fraction of cells in the Mic_np1, Mic_np2, and Mic_np3 subpopulations over time. (C) Venn plot of the mutual and unique marker genes for the Mic_np1, Mic_np2, and Mic_np3 subpopulations. (D) The volcano plot displays the differentially expressed genes between Mic_np clusters and Mic_pre clusters. The genes are colored according to the subpopulation with the higher expression. (E) The volcano plot displays the differentially expressed genes between Mic_np clusters and Mic_M1L clusters. The genes are colored according to the subpopulation with the higher expression. (F) Violin plot of the functional pathways revealed by Violin plot of the gene set variation analysis (GSVA) in the Mic_np1, Mic_np2, and Mic_np3 subpopulations. (G) Bar plot of the ratio of Arhgap45 + Iba1 + cells (green plus red) compared with DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6). (H) Bar plot of the ratio of Rgs10 + Iba1 + cells (green plus red) compared with DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6). (I) Bar plot of the ratio of Pkm + Iba1 + cells (green plus red) compared with DAPI + cells in a 500μm 2 area surrounding the infarction core (N=6).

Article Snippet: After the process of serum blocking, the antibody of the signature marker of each subpopulation was used for double staining, including Hpgd (the marker for Mic_home; Affinity; DF14850; 1:100), P2ry13 (the marker for Mic_pre1; allomone; APR-017; 1:100), Wsb1 (the marker for Mic_pre2; Affinity; DF12505; 1:100), Nfkbiz (the marker for Mic_M1L1; Affinity; DF12429; 1:100), Cd83 (the marker for Mic_M1L2; Affinity; AF5233; 1:400), Arhgap45(the marker for Mic_np1; proteintech; 14832-1-AP; 1:100), Rgs10 (the marker for Mic_np2;Affinity; DF4414; 1:100), and Pkm (the marker for Mic_np3; Affinity; DF5234; 1:100).

Techniques: Marker, Expressing, Functional Assay