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

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet: Unsupervised clustering and dimension reduction analysis of myeloid and NK cells reveals similar frequencies of subpopulations in AITD patients and controls (A) Non-T/non-B cells which encompass myeloid and natural killer (NK) cells were manually gated as CD45 + CD3-CD19-and then analyzed using the unsupervised clustering and dimension reduction programs PhenoGraph and X-shift to generate distinct clusters based on cell surface marker expression (CD15, CD1c, CD11b, HLA-DR, CD56, CD11c, CD14, and CD16). A UMAP plot was created using total non-B/non-T cells from concatenated adult AITD, adult healthy control, pediatric AITD, and pediatric healthy control samples with a minimum of 17,000 cell events. Seven unique cell subpopulations were identified. UMAP plots of each surface marker are displayed to demonstrate varying expression levels among the myeloid and NK cell subpopulations. (B) Heatmap of cell surface marker expression for identification of the 5 myeloid and 2 NK cell subsets identified using PhenoGraph/X-Shift. cDC1: conventional type 1 dendritic cell; cDC2: conventional type 2 dendritic cell. (C) Frequencies of myeloid and NK cell subsets in adult AITD (AA), adult healthy control (AH), pediatric AITD (PA), and pediatric healthy control (PH) subjects demonstrate normal frequencies in AITD patients compared to controls. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1 . Statistical significance determined by Mann-Whitney non-parametric unpaired Student's t tests.

Article Snippet: Mouse monoclonal anti-human CD11b (clone ICRF44) , Fluidigm , Cat#3209003B RRID: AB_2687654.

Techniques: Marker, Expressing, Control, MANN-WHITNEY

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-human CD11b (clone ICRF44) , Fluidigm , Cat#3209003B RRID: AB_2687654.

Techniques: Recombinant, Antibody Labeling, Immunopeptidomics, Mass Cytometry, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 2. CD11b is involved in the regulation of α-syn–mediated microglial migration. (A) Effect of fMLP receptor antagonist cyclosporin H and Ab against either α-syn or CD11b on migration of rat microglia toward purified rH α-syn aggregates or fMLP. Here 1.0 × 105 microglia were loaded onto each insert. (B) Effect of anti–α-syn or anti-CD11b Ab on migration of WT mouse microglia toward purified rH α-syn aggregates or fMLP. (C) Migration of CD11b−/−mouse microglia toward purified rH α-syn aggregates or fMLP. In A, B, and C, the transmigrated microglia were measured in the 96-well Boyden chambers using a CytoQuant assay kit. (D) Migration of rat primary microglia toward rat neuron-enriched cultures in which α-syn expression was intact, enhanced or knocked down. “Input” control reflects addition of 1.0 × 105 microglia directly to the well. (E) Migration of mouse WT or CD11b−/−microglia toward mouse or rat neuron-enriched cultures with or without an anti-CD11b Ab. Microglial migration in D and E was measured in the 24-well Boyden chambers. In A–E, n = 4. The Student t test was performed in E, whereas ANOVA followed by the Newman–Keuls multiple-comparisons test were performed in A–D. (F) Representative images showing rat primary microglial migration toward rat neurons with or without an anti-CD11b blocking Ab. Here 3.0 × 105 microglia were loaded onto each insert. (G) Quantitative analysis of microglia that overlapped neurons, counted in images as represented in F. n = 5, Student’s t test. In A–E and G, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding BSA or IgG controls; ##P < 0.01 and ###P < 0.001 compared as indicated. (Scale bar: 100 μm.)

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Migration, Purification, Expressing, Control, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 3. α-Syn aggregates directly bind to CD11b, which activates Nox2 to induce a migratory conformation of microglia. (A) In vitro binding assays. Myc-DDK– fused human CD11b was incubated with rH α-syn aggregates, and binding was detected via immunoblot for DDK and α-syn. Incubation of CD11b or α-syn alone served as controls. n = 3. (B) In vivo binding assays. Purified rH α-syn aggregates were mixed with WT or CD11b−/−microglial lysates to allow α-syn to react with CD11b, and the mixtures were further incubated in the IgG- or Ab-conjugated magnetic beads. Binding was detected via immunoblot for CD11b and α-syn. n = 3. (C) O2

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: In Vitro, Binding Assay, Incubation, Western Blot, In Vivo, Purification, Magnetic Beads

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 5. H2O2, a product of α-syn–activated Nox2, serves as a direct signal to regulate microglial directional migration on the interaction between α-syn and CD11b. (A) Microglial chemotaxis toward H2O2 with or without catalase (Cat) based on an under-agarose gel migration assay. n = 4. ANOVA, followed by the Newman–Keuls multiple-comparisons test. (B) Polarized microglial morphology and F-actin distribution after direct exposure of cells to 10 μM H2O2 for 30 min. (Scale bar: 10 μm.) (C) Effect of α-syn stimulation on extracellular H2O2 in WT mouse microglia with or without catalase, as measured by an Abcam kit. BSA served as a control (Ctr). (D) Extracellular H2O2 in stimulated CD11b−/−mouse microglial cultures. (E) Extracellular H2O2 in stimulated gp91phox−/−mouse microglial cultures. (F) Quantitative analysis of the changes in intracellular H2O2 concentration in WT mouse microglia. (G) Quantitative analysis of the changes in intracellular H2O2 concentration in WT mouse microglia pretreated with catalase overnight. In C, D, E, F, and G, ANOVA was performed comparing with the BSA-treated control. n = 4. (H) BSA- or catalase-preincubated microglia migrated toward BSA, fMLP, or α-syn. Assays were performed in the 96-well Boyden chambers using a CytoQuant kit, with 1.0 × 105 microglia loaded onto each insert. n = 4. ANOVA followed by the Newman–Keuls multiple-com- parisons test were performed. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding BSA-treated control. ###P < 0.001 as indicated.

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Migration, Chemotaxis Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 6. Lyn acts as an H2O2 sensor to regulate the phosphorylation of cortactin. (A) Exposure to either α-syn or H2O2 induced the phosphorylation of SFKs (pSFK) in WT primary mouse microglia. Tubulin (Tub) blots served as protein loading controls. (B) Lyn, an SFK family member, is phosphorylated in WT mouse microglia stimulated using either α-syn or H2O2. (C) Effect of DMSO (DM) or catalase (Cat) on α-syn– or H2O2-induced Lyn phosphorylation in WT mouse microglia. (D) Effect of DMSO (DM) or PP2 on α-syn– or H2O2-induced Lyn phosphorylation in WT mouse microglia. (E) Lyn phosphorylation (pLyn) in CD11b−/−

Article Snippet: To determine whether rH α-syn binds to Myc/DDK-tagged CD11b (OriGene Technologies), an equimolar mixture of these two proteins was incubated and further reacted with anti-Myc–tagged magnetic beads (MBL International).

Techniques: Phospho-proteomics

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