Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

doi: 10.64898/2026.03.15.711858

Figure Lengend Snippet: A , Schematic overview of the experimental design. WT-BMT and PAD4KO-BMT mice received either IgG isotype control or anti-Ly6G antibody (100 μg per mouse) by intraperitoneal injection every 3 days for 28 days, as indicated. B , Peripheral blood cell counts, and neutrophil and monocyte fractions (n=4-5). Neutrophils were determined by CD45 high Gr-1 high CD11b low and monocytes were determined by CD45 high Gr-1 low CD11b high by FACS analysis. C , Echocardiographic assessment 28 days after TAC (n=6 in each group). D , Representative fluorescent immunohistochemistry images of left ventricular tissue stained for neutrophil elastase (NE, green), citrullinated histone H3 (CitH3, red), and troponin I (yellow), with nuclear counterstaining by DAPI (blue). Arrowheads indicate NET-positive cells (NE + CitH3 + ). Scale bars, 50 μm. E , Quantification of the numbers of neutrophils (NE + ) and NETs (NE + CitH3 + ) per myocardial tissue area (n=4). F , Representative left ventricular sections stained for CD68 (green), CitH3 (red), troponin I (yellow) and DAPI (blue). Arrowheads indicate MET-positive cells (CD68 + CitH3 + ). Scale bars, 50 μm. G , Quantification of CD68 + macrophages and METs (CD68 + CitH3 + ) per tissue area (n=3-4). All data are presented as mean ± SEM. *P < 0.05 versus the corresponding IgG-treated group, † P < 0.05 versus IgG-treated WT-BMT mice, and ‡ P < 0.05 versus anti-Ly6G-treated WT-BMT mice as determined by one-way analysis of variance with Tukey’s post hoc analysis.

Article Snippet: Magnetic-activated cell sorting was performed on bone marrow cells using MACS MS columns (Miltenyi Biotec GmbH) with CD11b MicroBeads (130-097-142, Miltenyi Biotec GmbH) according to the manufacturer’s instructions.

Techniques: Control, Injection, Immunohistochemistry, Staining

Journal: The Journal of Clinical Investigation

Article Title: Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy

doi: 10.1172/JCI167417

Figure Lengend Snippet: ( A ) Brain tissue pieces were collected from the indicated brain regions and dissociated by mechanical disruption and enzymatic digestion. A CNS single-cell suspension was generated after Percoll separation. CD3 + T cells were positively selected and used for a CNS T cell QVOA. BrMCs and MG were isolated from the CD3 – fractions by CD11b + selection and by TMEM119 + selection, respectively. For human brains, CD11b + selection was performed to isolate BrMCs. BrMCs or MG at P0 (collected immediately after isolation) were used for purity and phenotype analysis and for RT-qPCR to measure proviral DNA and cell-associated RNA. BrMCs or MG at P1 were cultured 1–2 weeks ex vivo to allow the cells to recover and attach. P1 cells were used for the LRA study and the QVOA. ( B ) MG isolated from ART-suppressed, SIV-infected rhesus macaques were defined by TMEM119 staining (P1 MG) (scale bar: 100 μm) and ( C ) anti-TMEM119/anti-CD11b flow cytometry (P0 MG). ( D ) NHP MG proliferated ex vivo. ( E ) Total and integrated SIV DNA was detectable in isolated P0 MG ( n = 3). ( F ) SIV RNA was induced in isolated P1 MG 7 days after stimulation by the HDACi SAHA (500 nM), but was poorly induced by the canonical NF-κB agonist PEP005 (12 nM), the noncanonical NF-κB agonist AZD5582 (100 nM), or TNF-α (50 ng/mL). *** P < 0.001 compared with mock treatment, by 1-way ANOVA ( n = 3). ( G ) SIV RNA was recovered from the supernatant of NHP P1 MG cocultured with CEM174 ( n = 3). Data are presented as the mean ± SEM.

Article Snippet: For NHP samples, CD11b MicroBeads, NHP (catalog 130-091-100, Miltenyi Biotec) were used ( ).

Techniques: Disruption, Suspension, Generated, Isolation, Selection, Quantitative RT-PCR, Cell Culture, Ex Vivo, Infection, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Atf3 Integrates Lipid and Cytoskeletal Remodeling to Drive Macrophage Fusion

doi: 10.64898/2026.04.01.715652

Figure Lengend Snippet: (A-B) Representative confocal micrographs of IL-4 stimulated BMDMs stained against the membrane-localized integrin CD11b show numerous DAPI-stained nuclei within a single control BMDM (A-A’’). In contrast, Atf3 KO BMDMs form multilayered aggregates, with mononuclear cells clearly separated by cell membranes (B-B’’). White arrowheads indicate the positions of the cross-sections. (C-D) 3D confocal projection of z-stack images of control (C) BMDMs stained for F-actin (Phal) after three days of IL-4 stimulation shows prominent ruffling (R), while Atf3 KO BMDMs display scarce thin filopodia (Fl, arrow). White arrowheads indicate the positions of the cross-sections, which are presented below (C’, D’). (E-F) Electron microscopy images highlight long filopodia in control BMDMs treated with IL-4 for three days (e), compared to the shorter projections in Atf3 KO cells (F). (G-J) Compared to the rich cortical actomyosin network in control cells, visualized by staining for F-actin (Phal) (G, H, corresponding intensity heatmaps G’, H’) and phospho-Myosin (S19), actomyosin cables in Atf3 KO BMDMs (I, J) are concentrated beneath the plasma membrane and extend along the branches. (K-L) Confocal micrographs of BMDMs expressing Lifeact-EGFP reveal actin-rich podosome rings (K, asterisks), lamellipodia (K, arrowhead) and filopodia (K, arrow) in control cells while these structures are notably reduced in Atf3 KO BMDMs (l). (M-O) Representative confocal micrographs (M, N) and quantification (O) of IL-4 stimulated BMDMs showing increased WASP fluorescence intensity in Atf3 KO macrophages (N) compared with control cells (M). In contrast to the primarily nuclear signal in control BMDMs, Atf3 KO BMDMs display WASP signal in both nucleus and cytoplasm. (P) Representative western blot of IL-4 treated cells shows marked decrease of F-actin in Atf3 KO BMDMs compared to control . (Q-R) Representative western blots of WASP, phospho-Cofilin (p-Cofilin) and total Cofilin protein levels (Q) and quantification (R) in IL-4-stimulated BMDMs of the indicated genotypes. ATP5α was used as a loading control. (S-T) Representative confocal micrographs reveal differential localization and enrichment of Filamin A (FLNA) in the nucleus (arrow, N) of control (S, S’) cells and in perinuclear foci (PN) of Atf3 KO (T, T’) BMDMs stimulated by IL-4. Data information: Data represent means ± s.d.; **p<0.01, ****p<0.0001 (O, R). Statistical significance was determined using Mann-Whitney test. Each dot represents individual cell from three independent experiments (O, n = 3) or individual animal (R, n = 8 left, n = 15 right) Micrographs are z-projections of multiple confocal sections. DAPI labels nuclei. Scale bars: 10 µm (A-D, G-L, S, T), 50 µm (M, N), and 0.5 µm (E, F).

Article Snippet: Peritoneal Macrophages (PMs) were isolated from the peritoneal cavity of 8-10-week-old mice according to a previously described protocol ( Herb et al , 2019 ) using paramagnetic CD11b MicroBeads (MiltenyiBiotec #130-049-601).

Techniques: Staining, Membrane, Control, Electron Microscopy, Clinical Proteomics, Expressing, Fluorescence, Western Blot, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

doi: 10.1186/s13075-021-02663-z

Figure Lengend Snippet: High-fat diet increases monocytic-MDSC expansion during PTOA. A Gating strategy used to define the M-MDSC subpopulation (CD11b + Ly6C + Ly6G − cells) in mouse bone marrow. Following the initial FS/SC discrimination, the gate was set on CD11b + cells. After exclusion of doublets (not shown), live CD11b + cells were gated and Ly6C + and Ly6G + populations. B Cytospin images of M-MDSC cells from the bone marrow from LFD and HFD mice (at 16 weeks). Scale bar denotes 10 microns. C HFD increases the BM M-MDSC population in mice ( n = 4 in each group) by total number and percentage with concomitant suppression of CD8 + and CD4 + T-cells compared to LFD mice ( n = 6 in each group, at 16 weeks). D HFD increases the BM M-MDSC population in DMM mice ( n = 3 in each group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA Tukey’s multiple comparison test

Article Snippet: The following anti-human antibodies were purchased from Miltenyi Biotec (Germany): CD11b-APC (clone M1/70, # 130-113-793), HLA-DR–PE (clone AC122, #130-113-402), CD14-fluorescein isothiocyanate (FITC) (clone Tuk4, #130-113-146), CD15-APCvio770 (clone VIMC6, #130-104-992), CD33-viobright515 (clone REA775, #130-111-027), CD66b-PEvio770 (cloneREA306, #130-119-808), and their corresponding isotype controls, along with corresponding isotype controls.

Techniques: Comparison

Journal: Arthritis Research & Therapy

Article Title: Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

doi: 10.1186/s13075-021-02663-z

Figure Lengend Snippet: MDSCs are expanded in obese patients with post-traumatic osteoarthritis. A Demographic of the patient population. B Gating strategy used to define the M-MDSC subpopulation (CD11b + HLADR − CD14 + CD15 − cells). The M-MDSC population is expanded in C peripheral blood and D synovial fluid of obese ( BMI > 30; N = 10) compared to non-obese ( BMI < 30; N = 5) patients diagnosed with post-traumatic osteoarthritis. Two-tailed unpaired Student t -test

Article Snippet: The following anti-human antibodies were purchased from Miltenyi Biotec (Germany): CD11b-APC (clone M1/70, # 130-113-793), HLA-DR–PE (clone AC122, #130-113-402), CD14-fluorescein isothiocyanate (FITC) (clone Tuk4, #130-113-146), CD15-APCvio770 (clone VIMC6, #130-104-992), CD33-viobright515 (clone REA775, #130-111-027), CD66b-PEvio770 (cloneREA306, #130-119-808), and their corresponding isotype controls, along with corresponding isotype controls.

Techniques: Two Tailed Test

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 1. Cell surface expression of CD11b on different cell populations. The expression was quantified by flow cytometry using ICRF44 (A) and CBRM1/5 (B) antibodies. The latter only recognises the headpiece of CD11b in its active state. Data are presented in mean fluorescence intensity (MFI), closed symbols 77R/R donors, open symbols 77R/H-77H/H donors. The two groups were not statistically different. Bars indicate means. doi:10.1371/journal.pone.0057082.g001

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 2. Phagocytosis of hiC3b-coated fluorescent beads. The uptake by macrophages (A), PMNs (B), monocytes (C) and DCs (D) was quantified by flow cytometry and the data are represented as percentage of phagocytosis. Data are expressed as mean+/2SEM. Filled columns 77R/R individuals, open columns 77R/H-77H/H individuals. Paired T test was applied and the p values are indicated. doi:10.1371/journal.pone.0057082.g002

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Flow Cytometry

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 3. Rosetting assay. Percentage of rosettes formed by CFSE- labelled RBC-hiC3b with freshly isolated PMNs from donors carrying the susceptible allele (77H/H, open symbol) or the common allele (77R/R, closed symbol). One representative assay out of 3 independent experiments. doi:10.1371/journal.pone.0057082.g003

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Isolation

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 4. In vitro and in vivo PMN migration. (A) Migration of neutrophil cell lines through transwells seeded with mouse endothelial cells. PMNs migrated into the bottom chamber in response to MIP-2 were counted at different time points as indicated. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. Itgam2/2 and wild type C57BL/6 neutrophil cell lines were used as controls. CD11b- deficient PMNs, known to have weaker endothelial interactions, migrated faster than the C57BL/6 and the hCD11b expressing cell lines (p,0.001 at 60 mns and p,0.05 at 90 mns). Statistical analysis by Bonferroni’s multiple comparison test. (B) Time course of the migration of freshly isolated human 77R/R or 77R/H neutrophils through a HUVEC layer in response to MIP-2. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. (C, D) In vivo peritoneal migration of hCD11b-77R and hCD11b-77H PMN cells lines following i.p. injection of MIP-2 (C) and thioglycollate (D). The two hCD11b expressing PMN lines were labelled with DDAO or CFSE and adoptive transferred at a 1:1 ratio into C57BL/6 mice. Absolute numbers of labelled PMNs recovered from the peritoneum are shown. Data of one out of at least 3 independent experiments are presented. Bars indicate means. doi:10.1371/journal.pone.0057082.g004

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: In Vitro, In Vivo, Migration, Expressing, Comparison, Isolation, Injection

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 5. Cytokine response. Monocytes (A), DCs (B) were stimulated with 2 mg/ml and 10 mg/ml of TLR7/8 ligand (R848) respectively for 24 h. Cytokines quantified using a bead multiplex assay. Closed symbols: 77R/R cells, open symbols: 77R/H-77H/H cells. Each dot represents a single individual, bars denote means. No significant differences between the two CD11b genotypes. Statistical analysis by paired t test. (C, D) Modulation of TLR7/8-induced cytokine release by hiC3b-coated beads. Monocytes (C), DCs (D) were fed with hiC3b-coated beads one hour prior to R848 stimulation. The cytokine changes between the samples with and without CR3 pre-engagement with iC3b are shown with the p values indicated. Data are expressed as mean+/2SEM. The cytokine responses of 77R/R cells (black column) and 77R/H-77H/H cells (white columns) were not statistically different in paired assays. IL, interleukin; TNF-a, tumour necrosis factor alpha; IP-10, Interferon gamma-induced protein 10. doi:10.1371/journal.pone.0057082.g005

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Multiplex Assay