Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: CD11b is required for stretch-mediated changes in macrophage activation. (A) Representative Western blot of CD11b and GAPDH for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0% and 20% static and cyclic stretch (left). Quantification of average across three independent experiments for CD11b expression (right). Values were normalized to GAPDH and made relative to 0% stretch and unstimulated condition. (B) Representative immunofluorescence images (top) and quantification of relative Itgam gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Data relative to siControl condition. (C) Secretion of TNFα, IL6, and MCP1 for unstimulated and IFNγ/LPS stimulated macrophages treated with siControl or siCD11b and exposed to either 0% control, 20% static, or 20% cyclic stretch. Data normalized to a siControl and IFNγ/LPS treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Arg1 gene expression in IL4/IL13 stimulated and siControl or siCD11b treated BMDMs exposed to 0% control, 20% static, or 20% cyclic stretch. Data relative to 0% siControl condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (A, C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Gene Expression, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Modulation of CD11b by adhesion time regulates stretch-mediated macrophage inflammatory responses. (A) Phase contrast images (top) of macrophages following 4 hrs (left) and 24 hrs (right) of adhesion prior to stimulation and stretch. Fluorescence images (bottom) of macrophages labelled for CD11b (red), actin (green), and nuclei (blue) following 4 hrs (left) and 24 hrs (right) of culture. (B) Averaged relative median fluorescence intensity across three independent experiments of CD11b as measured by flow cytometry. Values normalized to 4 hrs adhesion condition. (C) Secretion of TNFα for unstimulated and IFNγ/LPS stimulated macrophages exposed to 0% and 20% cyclic stretch after 4 hrs (left) and 24 hrs (right) of adhesion. Values are normalized to a 0% stretch IFNγ/LPS internal control within each biological replicate. (D) Representative Western blots (left) and corresponding quantification for ARG1 (right) for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages allowed to adhere for 4 hrs prior to stimulation and stretch. Values were normalized to GAPDH and made relative to IL4/IL13 stimulated and 0% stretch conditions, respectively. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Fluorescence, Flow Cytometry, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Crosstalk between Piezo1 and CD11b mediates macrophage response to stretch. (A) Representative immunofluorescence images (left) and quantification of mean Piezo1-tdT intensity in unstimulated, IFNγ/LPS, and IL4/IL13 treated macrophages exposed to 0% control, 20% static, or 20% cyclic stretch. Data normalized to the 0% control condition. (B) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with siControl or siPiezo1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a siControl treated internal control exposed to 0% stretch within each biological replicate. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or Yoda1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a DMSO treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Piezo1 gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Gene expression is normalized to the siControl treated condition. (E) Relative Itgam, Itgb1, Itgb2 , and Itgb3 gene expression in unstimulated and siControl or siPiezo1 treated macrophages. Gene expression is normalized to the siControl treated condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Immunofluorescence, Control, Gene Expression, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Stretch-induced changes in macrophage activation require modulation of actin. (A) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0%, 20% static, and 20% cyclic stretch. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Data normalized to the unstimulated and 0% stretch control. (B) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to siControl or CD11b siRNA. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Values normalized to unstimulated and siControl condition. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Values are normalized to a DMSO, 0% stretch, and IFNγ/LPS stimulated internal control within each biological replicate. (D) Representative Western blot (left) and quantification of ARG1 expression in IL4/IL13 stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Expression is relative to GAPDH. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by paired t-test (A, B) and Student’s t-test (C, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Fluorescence, Control, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Summary of stretch mediated changes in macrophage function.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence and western blotting results showing the expression of relevant cytokines in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing ANGPT2 + , Tie2 + , and CD11b + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of ANGPT2 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Counts of TEMs (Tie2 + and CD11b + ) in each group (n = 9, one-way ANOVA and Šídák's multiple comparisons test). D Representative western blot showing the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 in the CIBT adjacent to the TM tissue in each group (normalized to β-actin expression). E Densitometric analyses of the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 (n = 3, one-way ANOVA and Šídák's multiple comparisons test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). The error bars represent the ± SDs. CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being washed with PBST, the slices were incubated with 488-conjugated goat anti-rabbit (1:2000, ab150077, Abcam, UK), 594-conjugated goat anti-mouse (1:2000, ab150116, Abcam, UK), and 647-conjugated donkey anti-goat (1:2000, ab150135, Abcam, UK) secondary antibodies at room temperature in a dark room for 2 h. The 647-conjugated CD11b antibody (0.5 μg, CL647-65229, proteintech, China) was used to directly label CD11b.

Techniques: Immunofluorescence, Western Blot, Expressing, Staining

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence results showing the expression of pro-angiogenesis factors in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and VEGFA + cells and in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of VEGF + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and IGF1 + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. D Counts of IGF1 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being washed with PBST, the slices were incubated with 488-conjugated goat anti-rabbit (1:2000, ab150077, Abcam, UK), 594-conjugated goat anti-mouse (1:2000, ab150116, Abcam, UK), and 647-conjugated donkey anti-goat (1:2000, ab150135, Abcam, UK) secondary antibodies at room temperature in a dark room for 2 h. The 647-conjugated CD11b antibody (0.5 μg, CL647-65229, proteintech, China) was used to directly label CD11b.

Techniques: Immunofluorescence, Expressing, Staining

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A–D Peritoneal macrophages from 6‐ to 10‐week‐old C57BL/6 mice were stained for the macrophage markers (A) F4/80, (B) CD11b, (C) MCM2 or (D) active DNA synthesis (EdU) 2 h after isolation. Scale bars: 20 μm. E–K Peritoneal macrophages were isolated from 6‐ to 10‐week‐old WT or SAMHD1 −/− (KO) mice. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP for 48 h and analysed for (G) infection, (H) MCM2 expression, (I) EdU incorporation (EdU was added at the time of infection), and co‐localisation between infection and (J) MCM2 protein or (K) active DNA synthesis (by EdU incorporation). (E, F) Random microscopic fields and plot profiles show an example of immunofluorescence intensity along infected cells. Scale bars: 10 μm. On average, 10 4 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ ( n ≥ 2, mean ± s.e.m.; (ns) non‐significant; **P ‐value ≤ 0.01, unpaired t ‐test).

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining, DNA Synthesis, Isolation, Infection, Expressing, Immunofluorescence, Imaging

Journal: Aging (Albany NY)

Article Title: Effects of magnetically targeted iron oxide@polydopamine-labeled human umbilical cord mesenchymal stem cells in cerebral infarction in mice

doi: 10.18632/aging.204540

Figure Lengend Snippet: In vivo anti-inflammatory effects of the HUMSCs and MIONs@PDA-MSCs. ( A ) Immunofluorescence analysis and quantification for M1 (CD11b) and M2 (CD206) macrophage markers in the cortex tissues of mice. scale bars=20 μm. ( B ) Western blot analysis and quantification of M1 (iNOS) and M2 (Arg-1) macrophage markers in the cortex tissues of mice. ( C ) Relative expressions of M1 ( IL-6, IL-1β and CD11b ) and M2 ( ARG-1, IL-10 and CD206 ) genes in the brain after treatment. * p < 0.05 vs. Sham group, # p <0 .05 vs. PBS group, & p <0 .05 vs. MSCs group, $ p <0 .05 vs. MIONs@PDA-MSCs group.

Article Snippet: After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes, which were then blocked with milk for 1 h. Afterwards, the proteins were labeled with the following primary antibodies overnight at 4° C: anti-MAP2, anti-CD206, anti-CD11b, anti-IBA-1, anti-β-actin (ProteinTech, Chicago, IL, USA) and anti-NeuN (Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vivo, Immunofluorescence, Western Blot