differentiation 11b cd11b  (Bio-Rad)

Journal: Frontiers in Immunology

Article Title: Microglial clock dysfunction during neuroinflammation impairs oligodendrocyte progenitor cell recruitment and disrupts neuroimmune homeostasis

doi: 10.3389/fimmu.2025.1620343

Figure Lengend Snippet: Microglial activation states oscillate across the circadian cycle under physiological conditions. (A) Experimental workflow. Mice were entrained under 12h:12h light:dark (LD) cycle for 14 days, followed by 1 day in constant darkness (DD). Brains were harvested every 4 hours across the circadian cycle for immunochemistry (IHC). (B) Confocal images of brain sections stained for CD11b (green) and IBA1 (red) at various circadian times (CT, hours). DAPI (blue) serves as a nuclear counterstain. Dashed boxes in lower magnification (Low mag.) images indicate regions enlarged in higher magnification (High mag.) panels. Dashed line outlines the cortex (Ctx) from the corpus callosum (CC). Images are shown as maximum intensity projections of three z-stack layers. Scale bars (white bars in bottom panels): 200 μm (Low mag.), 50 μm (High mag.). (C, D) Quantification of (B) , shown as mean ± SD. (C) Microglial density (IBA1& + cells/mm²) and branch number per IBA1& + microglia. n = 3 mice per CT. Each dot represents one mouse (density) or one cell (branching; ≥56 cells per CT from 3 mice). (D) Mean intensity of IBA1 and CD11b at each CT. Each dot represents one mouse. n = 3 mice per CT.

Article Snippet: After trypsinization determined by culture medium [1x HBSS (Gibco) + 2.5mM HEPES (Gibco) + 5.4g/L glucose (Sigma-Aldrich) + 100 units/mL penicillin + 100 μg/mL streptomycin (Gibco)], microglia were purified by cluster of differentiation molecule 11B (CD11b) beads (#130-097-142, Miltenyi) and followed the magnetic-activated cell sorting (MACS) protocol according to Miltenyi’s instructions.

Techniques: Activation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Microglial clock dysfunction during neuroinflammation impairs oligodendrocyte progenitor cell recruitment and disrupts neuroimmune homeostasis

doi: 10.3389/fimmu.2025.1620343

Figure Lengend Snippet: Microglial activation is further enhanced during neuroinflammation. (A) Experimental workflow illustrating the induction of neuroinflammation. Mice received an intraperitoneal (i.p.) injection of either PBS (control) or lipopolysaccharide (LPS) at circadian time (CT) 04. Brains were harvested three days post-injection at CT06. (B) Confocal images showing CD11b (green) and IBA1 (red) immunostaining. DAPI (blue) serves as a nuclear counterstain. Dashed boxes in lower magnification (Low mag.) images indicate regions enlarged in higher magnification (High mag.) panels. Dashed lines outline the cortex (Ctx), corpus callosum (CC), and hippocampus (Hippo). Images are maximum intensity projections of three confocal z-planes. Scale bars (white bars in top panels): 200 μm (Low mag.), 50 μm (High mag.). (C) Quantification of data from (B) , presented as mean ± SD. n = 2 mice per condition. Each dot represents one microglial cell (branch number; ≥31 cells per condition from 2 mice) or one hemi-cerebrum (mean intensity). Asterisks indicate statistical significance (Student’s t-test). ** P < 0.01; **** P < 0.001.

Article Snippet: After trypsinization determined by culture medium [1x HBSS (Gibco) + 2.5mM HEPES (Gibco) + 5.4g/L glucose (Sigma-Aldrich) + 100 units/mL penicillin + 100 μg/mL streptomycin (Gibco)], microglia were purified by cluster of differentiation molecule 11B (CD11b) beads (#130-097-142, Miltenyi) and followed the magnetic-activated cell sorting (MACS) protocol according to Miltenyi’s instructions.

Techniques: Activation Assay, Injection, Control, Immunostaining

Journal: Frontiers in Immunology

Article Title: Microglial clock dysfunction during neuroinflammation impairs oligodendrocyte progenitor cell recruitment and disrupts neuroimmune homeostasis

doi: 10.3389/fimmu.2025.1620343

Figure Lengend Snippet: Neuroinflammation alters microglial clock rhythmicity. (A) Experimental workflow illustrating the induction of neuroinflammation and preparation of microglia at various circadian times (CTs). Mice received an intraperitoneal (i.p.) injection of either PBS (control) or lipopolysaccharide (LPS, neuroinflammation) at CT04. Brains were harvested every 4 hours across the circadian cycle on day 3 post-injection. (B-D) qRT-PCR analysis showing the effects of neuroinflammatory conditions on the expression of (B) microglial activation markers ( Iba1 and Itgam ) and an irrelevant control gene ( Rpl30 ), (C) core clock genes ( Bmal1 and Per1 ), and (D) pro-inflammation chemokines ( Ccl3 and Ccl5 ). Expression values are normalized to a control gene ( Rps13 ) and presented as mean ± SD. Black and red lines represent control and neuroinflammatory microglia, respectively. n = 3 mice per CT. Asterisks indicate statistical significance (Student’s t-test). * P value < 0.05; ** P value < 0.01; *** P value < 0.005.

Article Snippet: After trypsinization determined by culture medium [1x HBSS (Gibco) + 2.5mM HEPES (Gibco) + 5.4g/L glucose (Sigma-Aldrich) + 100 units/mL penicillin + 100 μg/mL streptomycin (Gibco)], microglia were purified by cluster of differentiation molecule 11B (CD11b) beads (#130-097-142, Miltenyi) and followed the magnetic-activated cell sorting (MACS) protocol according to Miltenyi’s instructions.

Techniques: Injection, Control, Quantitative RT-PCR, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Dark Microglia Are Abundant in Normal Postnatal Development, where they Remodel Synapses via Phagocytosis and Trogocytosis, and Are Dependent on TREM2

doi: 10.1101/2024.10.15.618087

Figure Lengend Snippet: A-B: Using immunocytochemical transmission electron microscopy, dark microglia in development in the ventral hippocampus CA1 strata radiatum and lacunosum-moleculare of P10 C57BL/6J mice were not found to express TMEM119. A: Dark microglia (dm) process ensheathing a blood vessel (bv). A typical microglia (m) cell body stained for TMEM119 is also seen contacting a nearby blood vessel. B: Higher magnification view of the dark microglia process showing direct juxtaposition (arrowhead) with a typical microglia process stained for TMEM119. This dark microglia process is also touching (arrow) an axon terminal (t) making a synapse onto a dendritic spine (s). Both the dark and typical microglia processes are additionally associated with an extracellular space pocket containing partially degraded cellular elements or debris (cd). C-D: Example of dark microglia stained for CD11b observed in the ventral hippocampus CA1 strata radiatum and lacunosum-moleculare of P10 C57BL/6J mice using transmission electron microscopy. C: The dark microglia process is seen surrounding a blood vessel and juxtaposing a dendrite (d). D: Higher magnification view of the process showing CD11b staining on its distal extremities (arrowheads), where touching or encircling neuronal elements, such as dendritic spines (s) and axon terminals (t). E-F: Example of dark microglia stained for CLEC7a obtained using scanning electron microscopy in P15 C57BL/6J mice. F: Higher magnification view of the dark microglia with immunoreactivity for CLEC7a (arrowheads). G-H: Examples of dark microglia stained for LPL observed in the ventral hippocampus CA1 of P10 C57BL/6J mice using transmission electron microscopy. G: A dark microglia wrapped around a blood vessel display positive immunostaining for LPL in its processes (arrowhead). H: Dark microglial processes immunopositive for LPL (arrowheads) near a dendritic spine (s) and axon terminal (t).Representative electron micrographs. Scale bars are indicated on the electron micrographs. bv=blood vessel; cd=cellular debris; dm=dark microglia; m=microglia; s=dendritic spine; t=axon terminal. I-M: Imaging mass cytometry (IMC) I: Unsupervised cluster (C_1-16) Phenographs obtained from IMC analysis of hippocampus cornu ammonis (CA)1 at P14, visualised on a t-SNE plot. J: Representative picture of the different spatial distribution of C_1-16 via pseudocolouring of single hippocampal cells. K: Heatmap showing the relative mean expression of the 18 markers used in the IMC analysis. Marker attribution identified putative cell types for the following clusters: dark microglia (C_5), typical microglia (C_15), MBP + myelin structures (C_1), neuronal clusters (C_4,12,13), CD31 + IgM+ blood vessels (C_7), GFAP + astrocytes (C_6,8), CD11c + dendritic cells (C_9), KI67 + proliferating cells (C_14). Some clusters (C_2,3,10,11,16) could not be unequivocally assigned to a specific cell type. L: Representative picture showing different spatial distribution of the C_5 dark microglia, C_15 typical microglia and C_8 astrocytes in relation to gephyrin (green) and VGAT (red) expression. Nuclei are in blue. M: Heatmap displaying spatial interactions between Phenograph-derived clusters as revealed by neighbourhood analysis. Rows 5 and 15 are boxed, showing the cell clusters (columns) in the neighbourhood of C_5 (dark microglia) and C_15 (typical microglia). Colors indicate the prevalence of cell-type interactions across the region of interest, with blue squares representing avoidance and red representing positive interactions.

Article Snippet: Immunostaining with specific antibodies against transmembrane protein 119 (TMEM119) (abcam, #ab209064), cluster of differentiation molecule 11b (CD11b) (AbD Serotec, #MCA711GT), C-type lectin domain family 7 member A (CLEC7a) (Invivogen, mabg-mdect), lipoprotein lipase (LPL) (abcam, #ab21356) and triggering receptor expressed on myeloid cells 2 (TREM2) (R&D Systems, #AF1729), was performed as described in .

Techniques: Transmission Assay, Electron Microscopy, Staining, Immunostaining, Imaging, Mass Cytometry, Expressing, Marker, Derivative Assay

Journal: bioRxiv

Article Title: Dark Microglia Are Abundant in Normal Postnatal Development, where they Remodel Synapses via Phagocytosis and Trogocytosis, and Are Dependent on TREM2

doi: 10.1101/2024.10.15.618087

Figure Lengend Snippet:

Article Snippet: Immunostaining with specific antibodies against transmembrane protein 119 (TMEM119) (abcam, #ab209064), cluster of differentiation molecule 11b (CD11b) (AbD Serotec, #MCA711GT), C-type lectin domain family 7 member A (CLEC7a) (Invivogen, mabg-mdect), lipoprotein lipase (LPL) (abcam, #ab21356) and triggering receptor expressed on myeloid cells 2 (TREM2) (R&D Systems, #AF1729), was performed as described in .

Techniques: Saline

Journal: PLOS ONE

Article Title: Oligodendroglia-to-pericyte conversion after lipopolysaccharide exposure is gender-dependent

doi: 10.1371/journal.pone.0308132

Figure Lengend Snippet: (A) Immunostaining for GFAP was performed in the cortex of the male group, the female group, the male + LPS group and the female + LPS group. (B) Immunostaining for CD11b was conducted in the cortex of across the groups. Scale bar: 20 μm.

Article Snippet: For immunofluorescence staining, the sections underwent overnight incubation at 4°C with primary antibodies that target specific proteins: cluster of differentiation 13 (CD13, GTX75927, Genetex), platelet-derived growth factor receptor β (PDGFRβ, AF1042, R&D Systems; 14-1402-82, ThermoFisher), neuronal nuclei (NeuN, 266004, Synaptic Systems), GFAP (D262817, Sangon Biotech), cluster of differentiation 11b (CD11b, 557394, BD Bio-sciences), and ionized calcium-binding adapter molecule 1 (Iba-1, 019–19741, WAKO).

Techniques: Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction

doi: 10.1186/s12974-024-03107-6

Figure Lengend Snippet: TBI exacerbates HFD-induced increases in VAT inflammatory responses. A The effect of HFD and TBI on VAT weight (g/g BW) at 28 dpi. B TBI-HFD mice display a significant increase in CD45 + and CD11b + C ATM/myeloid cells when compared to Sham-HFD counterparts. Representative dot plot for % CD45 + /CD11b + ATM/myeloid cells D . TBI-HFD significantly increased phagocytotic (Bead + CD11b + myeloid) cell number when compared to Sham-HFD counterparts E . Neither TBI nor HFD impacted the number of CD11b − lymphocyte cell population F . Sham-HFD mice display a significant increase in VAT expression of TNF-α , NLRP3 , p22 phox , and IL-10 , when compared to Sham-SD counterparts G . TBI-HFD mice exhibit a significant increase in VAT expression of IL-1β and NLRP3 , when compared to TBI-SD counterparts and notably compared to Sham-HFD in the case of NLRP3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs Sham-SD; + + + + p < 0.0001 vs TBI-SD; ## p < 0.01 vs Sham-HFD. N = 6–7 per group. Data expressed as mean ± SEM

Article Snippet: A magnetic bead-conjugated anti-cluster of differentiation 11b (CD11b) was used to isolate microglia/macrophages from ipsilateral (injured hemisphere) cortical and hippocampal tissue using Miltenyi MACS Separation Technology (Miltenyi Biotec, Auburn, CA) as per manufacturer’s instructions.

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction

doi: 10.1186/s12974-024-03107-6

Figure Lengend Snippet: TBI and HFD selectively interact to amplify brain microglia transcriptome changes that indicate the activation of inflammation pathways . A Analysis on isolated CD11b + cells (microglia) ran on a nCounter Mouse Glial Panel (Nanostring Technologies; > 750 genes related to glial functions and metabolism) using a general heat map generated by the Morpheus unsupervised hierarchical clustering reveals that TBI is the primary driver of changes in gene expression; HFD has lesser yet detectable effects and a consistent gradient of changes is observed with the following group order: Sham-SD, Sham-HFD, TBI-SD, TBI-HFD. B Microglia-phenotype specific heat maps identify that TBI-SD mice display a consistent downregulation of genes associated with the homeostatic microglia and microglia markers phenotype, when compared to Sham-SD counterparts, at 90 dpi. In contrast, TBI-SD mice display an upregulation of genes associated with disease-associated microglia (DAM), pro-inflammatory-associated, and autophagy microglial phenotypes, when compared to Sham-SD counterparts. Overall, our data suggests that HFD consistently amplifies these effects. N = 3 pooled samples/group

Article Snippet: A magnetic bead-conjugated anti-cluster of differentiation 11b (CD11b) was used to isolate microglia/macrophages from ipsilateral (injured hemisphere) cortical and hippocampal tissue using Miltenyi MACS Separation Technology (Miltenyi Biotec, Auburn, CA) as per manufacturer’s instructions.

Techniques: Activation Assay, Isolation, Generated, Gene Expression

Journal: Journal of Neuroinflammation

Article Title: Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction

doi: 10.1186/s12974-024-03107-6

Figure Lengend Snippet: TBI is the primary factor of change in the flowthrough cellular transcriptome. nCounter Mouse Glial Panel analysis was performed in the isolated CD11b − populations (neurons, astrocytes, oligodendrocytes, etc.). Only the genes that showed evidence of significant changes (p < 0.05 in at least one of the two-way ANOVA outcomes; n = 121 genes) were included and the resulting heat map displayed a consistent gradient of changes across groups that matched the clustering previously observed in the microglia (Sham-SD, Sham-HFD, TBI-SD, TBI-HFD). TBI-SD mice displayed a significant decrease in neuronal genes, with a concurrent increase in genes associated with oligodendrocyte responses, when compared to Sham-SD counterparts. N = 3 pooled samples/group. Data expressed as mean ± STDEV

Article Snippet: A magnetic bead-conjugated anti-cluster of differentiation 11b (CD11b) was used to isolate microglia/macrophages from ipsilateral (injured hemisphere) cortical and hippocampal tissue using Miltenyi MACS Separation Technology (Miltenyi Biotec, Auburn, CA) as per manufacturer’s instructions.

Techniques: Isolation