cd11b expression  (MedChemExpress)

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: Administration of ApoSQ-CAF CM reduces TAM density and M2 TAM fraction in primary tumors via WISP-1. ( a ) Schematic of experimental design and treatment groups. Starting two days after subcutaneous implantation of 344SQ cells into syngeneic (129/Sν) mice, intratumoral injections of conditioned medium from CAFs only (CAF CM), exposed to ApoSQ (ApoSQ-CAF CM) CAF CM, ApoSQ-CAF CM combined with anti-WISP-1, or ApoSQ-CAF CM combined with control IgG were administered three times per week for six weeks (n = 6 mice per group). Mice were necropsied at the end of the 6-week treatment period. ( b , c ) Left : Immunofluorescent staining of the pan-macrophage marker CD11b (red) and F4/80 (red), along with DAPI (blue), in central and marginal regions of primary tumors. Images were acquired at ×40 magnification. Scale Bar = 100 μm. Right : Quantitation of CD11b + and F4/80 + TAM density. ( d , e ) Upper : Immunofluorescent staining of primary tumor sections showing M2 TAM Markers Arg1 (green) and CD206 (green), along with the pan-macrophage marker CD11b (red). Original magnification: ×40. Scale bars = 100 μm. Lower : Quantitation of Arg1 + and CD206 + TAM (M2) density ( left ) and the fraction of M2 TAMs ( right ) in primary tumors. The M2 TAM fraction was determined by the percentage of M2 TAMs within CD11b + TAMs. NS, not significant; ** P < 0.01, *** P < 0.001 compared to CAF CM or as indicated; ### P < 0.001 compared to CAF CM, Analysis of variance with Tukey's post hoc test. The data are from one experiment representative of three independent experiments with similar results ( b and c left ; d and e upper ). The data are represented as the means ± standard errors from three mice per group ( b and c right ; d and e lower ).

Article Snippet: For macrophage enrichment, TAMs expressing CD11b were isolated using CD11b MicroBeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's instructions.

Techniques: Control, Staining, Marker, Quantitation Assay

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: ApoSQ-CAF CM promotes M2-to-M1 TAM reprogramming and activates STAT1 in M2 TAMs via WISP-1. The experimental design was described in Fig. a. ( a ) Heatmap showing differentially expressed genes encoding M1 and M2 marker-related molecules in isolated CD11b + TAMs from primary tumors (left). Red: high expression; blue: low expression. Relative expression of selected genes from PCR array profiling of macrophage polarization markers (right). Log2 fold-change values (ApoSQ-CAF CM vs. CAF CM). ( b ) qRT-PCR analysis of relative mRNA levels of M2 markers ( Arg1 , Cd206 , Cd163 , Il4 , Il10 , Tgfβ1 ), and M1 markers ( Tnfα , Cd80 , MhcII , Nos2 , Ifng , and Il12p40 ) in isolated CD11b + TAMs from primary tumors. NS: not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, Analysis of variance with Tukey's post hoc test. ( c ) Immunoblot analysis of Arg1, CD206, iNOS, and CD16/32 in isolated CD11b + TAMs from primary tumors. ( d , e ) Flow cytometry analysis of the population of M1 TAMs (MHCII + and CD80 + ) and M2 TAMs (CD163 + and CD206 + ) in CD11b + TAMs from primary tumors. Mean fluorescence intensity (MFI) values ( right ). ( f ) Upper: Representative flow cytometry plots in CD11b + TAMs. Lower : TAM ratio (CD163 + /MHCII + TAMs). ( g-k ) Flow cytometry analysis of the population of M2 macrophages ( g ), Tregs ( h ), M1 macrophages ( i ), CD8 + T cells ( j ), and CD4 + T cells ( k ). Tumor-infiltrating immune cells were stained with antibodies against CD45, CD11b, CD3, CD4, CD8, FoxP3, MHCII, and Ly6C. Absolute number of each cell type was counted using flow cytometry. ( a , d-k ) NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. ( a - k ) The data are from three replicates per condition, with cells pooled from three mice per replicate. ( l, n ) Representative confocal images of primary tumor sections stained with an anti-phosphorylated STAT1 (red), anti-CD206 antibody (green), anti-iNOS antibody (green), and DAPI (blue). Original magnification: ×40. Scale bars = 100 μm. ( m , o ) Quantification of phosphorylated STAT1 + cells among CD206 + cells and iNOS + cells. NS, not significant; *** P < 0.001, Analysis of variance with Tukey's post hoc test. The data are from one experiment representative of three independent experiments with similar results ( a , d and e left ; c , l , n ; f upper ) or from three independent experiments (mean ± standard error: a , d and e right ; b, g-k , m , o ; f lower ).

Article Snippet: For macrophage enrichment, TAMs expressing CD11b were isolated using CD11b MicroBeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's instructions.

Techniques: Marker, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Fluorescence, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: Administration of rWISP-1 reduces TAM density, decrease the M2 fraction and marker expression, and activates STAT1 in M2 TAMs. The experimental design was described in Supplementary a. Where indicated, rWISP-1 (12.5 and 25 μg/kg) was administered intratumorally three times a week for 6 weeks starting 2 days after subcutaneous implantation of 344SQ cells into syngeneic (129/Sν) mice (n = 6 mice per group). Mice were necropsied 6 weeks later. ( a , b ) Upper : Immunofluorescent staining of primary tumor sections showing M2 TAM Markers Arg1 (green) and CD206 (green), along with the pan-macrophage marker CD11b (red). Original magnification: ×40. Scale bars = 100 μm. Lower : Quantitation of Arg1 + and CD206 + TAM (M2) density ( left ) and the fraction of M2 TAMs ( right ) in primary tumors. The fraction of M2 TAMs were determined by the percentage of M2 TAMs within CD11b + TAMs. ( c ) qRT-PCR analysis of relative mRNA levels of M2 markers ( Arg1 , CD206 , CD163 , IL-4 , IL-10 , TGF-β1 ), and M1 markers ( TNFα , CD80 , MhcII , NOS2, Ifng, and IL-12 p40 ) in isolated CD11b + TAMs from primary tumors. ( d ) Immunoblot analysis of Arg1, CD206, iNOS, and CD16/32 in isolated CD11b + TAMs from primary tumors. ( e, g ) Representative confocal images of primary tumor sections stained with an anti-phosphorylated STAT1 (red), anti-CD206 antibody (green), anti-CD86 antibody (green), and DAPI (blue). Original magnification: ×40. Scale bars = 100 μm. ( f , h ) Quantification of phosphorylated STAT1 + cells among CD206 + cells and CD86 + cells. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to Vehicle or as indicated; ### P < 0.001 compared to Vehicle, Analysis of variance with Tukey's post hoc test. The data are from one experiment representative of three independent experiments with similar results ( a and b upper ; d , e , g ). The data are represented as the means ± standard errors from three mice per group ( a and b lower ; c , f , h ).

Article Snippet: For macrophage enrichment, TAMs expressing CD11b were isolated using CD11b MicroBeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's instructions.

Techniques: Marker, Expressing, Staining, Quantitation Assay, Quantitative RT-PCR, Isolation, Western Blot

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: The myeloid compartment in B16.F10 melanoma shifts toward suppressive phenotypes as tumors develop (A) Flow cytometry quantification of the percentage of DCs (CD11c+) and phagocytes (CD11b+CD11c-) within CD45 + cells. (B) Flow cytometry quantification of MDSCs populations (moDCs; CD11b + CD11c + Ly6C + , M-MDSCs; CD11b + CD11c − Ly6C + , and G-MDSCs; CD11b + CD11c − Ly6G + ) within CD45 + cells. (C) Flow cytometry quantification of the expression levels of immune-modulatory markers PD-L1, FasL, ARG1, and NOS2 by moDCs, M-MDSCs, and G-MDSCs. (D) Representative CFSE plots for CD8 T cell proliferation after culture alone, co-culture with tumor-derived CD11b + Ly6C − cells, or co-culture with a mix of tumor-derived moDCs and M-MDSCs. Black bar highlights the gated proliferated cells. (E) Flow cytometry quantification of the percentage of proliferating CD8 and CD4 cells cultured alone, co-cultured with tumor-derived CD11b + Ly6C − cells, or a mix of moDCs and M-MDSCs. (F) Representative CFSE plots for CD4 and CD8 T cell proliferation after co-culture with pre-sorted, tumor-derived moDCs or M-MDSCs. Black bar highlights the gated proliferated cells. (G) Quantification of T cell suppression following incubation with pre-sorted, tumor-derived moDCs and M-MDSCs compared to T cells cultured alone. Data are mean ± SEM; ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (A–C) Mixed effect analysis with a Tukey’s post hoc test. (E and G) One-way ANOVA with a Tukey’s post hoc test. (A and B) n = 5 mice for day-5 and day-9 tumors and n = 6 for day-11 tumors, from two independent experiments comparing each cell type at day 5, 9, or 11 time points with the day 0 time point. (C) n = 8 mice for day 5 tumors and n = 6 mice for day 11 tumors from two independent experiments. (E) n = 3 and (G) n = 2 mice performed in duplicate from two different experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Derivative Assay, Cell Culture, Incubation

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: Distribution of CD47 and SIRPα expression across the TME (A) Clustering of stromal populations identified in B16.F10 melanomas and matched draining lymph nodes analyzed from data previously published by Davidson et al. (B) Expression of CD47 and its cognate receptor, SIRPα, distributed across stromal clusters. (C) Violin plots highlighting widespread CD47 but restricted SIRPα expression across stromal subsets. (D) Flow cytometry quantification of CD47 expression at the protein level in T cells, (immunomodulatory) CAF 1, (myofibroblast) CAF 2, myeloid cells, endothelial cells (CD31 + ), and B16.F10 tumor cells. (E) Representative confocal image of a day 11 B16.F10 melanoma showing myeloid populations. Arrows indicate CD11b+Ly6C + SIRPα+ cells. Insets show zoom of selected ROI. Arrowheads depict cells positive for CD11b but negative for Ly6C and SIRPα. DAPI (gray), CD11b (red), Ly6C (green), SIRPα (blue). Scale bar is 50μm. Data are mean ± SEM; ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (D) One-way ANOVA with a Dunnett post hoc test. (D) n = 3 replicates from two independent experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet:

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Purification, Recombinant, Modification, Red Blood Cell Lysis, Cell Isolation, Staining, ATP Assay, Quantitation Assay, Software