cd86 (clone bu63) antibody  (SouthernBiotech)

Journal: Journal of biomedical materials research. Part A

Article Title: DENDRITIC CELL RESPONSES TO SELF-ASSEMBLED MONOLAYERS OF DEFINED CHEMISTRIES

doi: 10.1002/jbm.a.32487

Figure Lengend Snippet: Dendritic cells treated with different SAMs exhibited differential expression levels of DC maturation markers. Dendritic cells matured with LPS exhibited higher expression of CD80, CD83 or HLA-DQ compared to iDCs. Dendritic cells treated with OH, COOH or NH2 SAMs increased expression of CD80, CD83 or CD86 as compared to iDCs, but to a lower extent than DCs treated with LPS as observed with CD80 expression. In contrast, DCs treated with CH3 SAMs had higher expression of HLA-DQ as compared to iDCs. This experiment was performed using six independent donors in triplicate. Data represented as treatment control ratios for each treatment revealed statistical significance of the findings; mean ± SEM, of pooled average ratios for each donor from n=6 donors; ‘+’ indicates increase over iDC, differences between SAMs in words, p≤0.05.

Article Snippet: Loosely adherent cell fractions containing DCs for iDCs, mDCs or DCs treated with different SAMs were collected and resuspended in Hank’s HEPES buffer (120 mM NaCl, 10 mM KCl, 10 mM MgCl 2 , 10 mM glucose, 30 mM HEPES) (all from Sigma) containing 1% Human Serum Albumin (HSA) (EMD Biosciences, San Diego, CA) and 1.5 mM CaCl 2 (Sigma) and stained with saturating concentrations of fluorescently-labeled mouse anti-human monoclonal antibody against CD14 (clone UCHM1; IgG2aκ), CD40 (clone B-B20; IgG1κ), CD80 (clone BB1; IgMκ), CD86 (clone BU63; IgG1κ), CD19 (clone SJ25-C1; IgG 1 ), CD14 (clone UCHM-1; IgG2a) (all from Southern Biotech, Birmingham, AL), CD83 (clone HB15a; IgG2b) (IO Test Immunotech Beckman Coulter, Marseille, France) HLA-DQ (clone TU169; IgG2aκ), HLA-DR (clone TU36; IgG2aκ), CD3 (clone SK7; IgG1κ), CD24 (clone ML5; IgG2aκ) (all from Becton Dickinson Pharmingen, San Diego, CA), CD1c (clone AD5–8E7; IgG2a) (Miltenyi Biotec, Auburn, CA), or cytotoxic T lymphocyte associated antigen receptor-4 (CTLA-4-Ig) (clone 48815; IgG 2b ) (R&D Systems, Minneapolis) while maintained on ice and in the dark.

Techniques: Quantitative Proteomics, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses

doi: 10.3389/fimmu.2022.842241

Figure Lengend Snippet: Anti-mouse antibodies.

Article Snippet: Next, moDC were stained with an anti-CD86 FITC conjugated antibody (clone BU63) (ImmunoTools, Friesoythe, Germany) for 30 minutes at 4°C.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses

doi: 10.3389/fimmu.2022.842241

Figure Lengend Snippet: Adjuvant inclusion in GM3 liposomes does not alter binding properties of liposomes and TLRL-inclusion induces moDC maturation. (A) Liposomes were coated on an ELISA plate and the organic solvent was evaporated overnight. Subsequently, binding to WT or mutant CD169 was determined. Indicated is the average ± SEM of three independent experiments, performed in triplicate. Significance was compared to the non-GM3-containing counterpart. (B) Liposomes were incubated with moDC at 37°C, that were untreated or pretreated with a blocking antibody 15 minutes prior to liposome incubation. Indicated is the average DiD-signal ± SEM ( n = 4-8). (C) As in A, indicated is the average ± SEM of a technical triplicate. Significance was compared to the non-GM3-containing counterpart. (D) Liposomes were incubated with moDC (that were pretreated with type I IFN) at 37°C, that were untreated or pretreated with a blocking antibody 15 minutes prior to liposome incubation. Indicated is the average DiD-signal ± SEM ( n = 4-6). (E) MoDC were non-treated or incubated with a blocking antibody for 15 minutes and subsequently incubated with liposomes for 45 minutes at 37°C. Next, unbound liposomes were washed away and cells were incubated for 15-18h at 37°C, after which the cells were stained and the expression of CD86 was determined by flow cytometry analysis. Indicated is the average geometric mean fluorescent intensity (GMFI) of CD86 ± SEM ( n = 4-6). (F) as in E, but cells were pretreated with type I IFN (100 IU/mL). Indicated is the average GMFI of CD86 ± SEM ( n = 6-8). *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.

Article Snippet: Next, moDC were stained with an anti-CD86 FITC conjugated antibody (clone BU63) (ImmunoTools, Friesoythe, Germany) for 30 minutes at 4°C.

Techniques: Adjuvant, Liposomes, Binding Assay, Enzyme-linked Immunosorbent Assay, Solvent, Mutagenesis, Incubation, Blocking Assay, Staining, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

doi: 10.1038/srep18765

Figure Lengend Snippet: ( A ) mRNA expression profile of pro-( CXCL8 , CD80 , IL1B , TNF , CCR7 , CCL2 , IL6 ) and anti-inflammatory ( CD163 , MRC1 , VCAN ) macrophage markers, by real-time PCR, 20 h after 10 Gy. Graphs represent mRNA expression of irradiated macrophages compared to non-irradiated ones (dotted line) (at least n = 7 per marker). β-actin was used as housekeeping gene. Wilcoxon signed rank test was used to compare the median of each dataset against a hypothetical median value of 1. ( B ) Expression of a monocyte/macrophage lineage (CD14), pro-(HLA-DR and CD86) and anti-inflammatory (CD163) macrophage markers was determined, by flow cytometry, 20 h after irradiation (at least n = 6 per marker). Paired t -test was used for statistical analysis. ( C ) Levels of pro-(IL-6, IL-12/IL-23(p40)) and anti-inflammatory (TGF-β1 and IL-10) cytokines were determined in macrophage CM ( n = 9) by ELISA, 20 h after irradiation. Data was normalized to protein concentration. Wilcoxon matched pair test was used for statistical analysis. * P < 0.05, ** P < 0.01. Median is represented by the horizontal line inside the box plots, while the average is indicated by a “+”. In IL-6 and IL-10 graphics outliers are also indicated.

Article Snippet: Stainings with anti-human CD14-APC (clone MEM-18), HLA-DR-PE (MEM-12), CD86-FITC (clone BU63) (Immunotools) and CD163-PE (clone GHI/61) (R&D Systems) antibodies were performed in the dark for 30 min. After additional washing steps, macrophages were fixed for 15 min in 4% paraformaldehyde (PFA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Irradiation, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Scientific Reports

Article Title: Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

doi: 10.1038/srep18765

Figure Lengend Snippet: Non-irradiated and 10 Gy irradiated macrophages ( n = 6) were stimulated, for 20 h, with LPS (100 ng/ml) and IFN-γ (20 ng/ml) towards a pro-inflammatory, or with M-CSF (10 ng/ml) and IL-10 (20 ng/ml) towards an anti-inflammatory phenotype. ( A ) Expression of monocyte/macrophage lineage (CD14), pro-(HLA-DR and CD86) and anti-inflammatory (CD163) macrophage markers was determined by flow cytometry. ( B ) Macrophage CM levels of pro-(TNF-α, IL-6, IL-12/IL-23(p40)) and anti-inflammatory (TGF-β1 and IL-10) cytokines were determined by ELISA. Data was normalized to protein concentration. Paired t -test and one-way ANOVA were used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001. Median is represented by the horizontal line inside the box plots, while the average is indicated by a “+”.

Article Snippet: Stainings with anti-human CD14-APC (clone MEM-18), HLA-DR-PE (MEM-12), CD86-FITC (clone BU63) (Immunotools) and CD163-PE (clone GHI/61) (R&D Systems) antibodies were performed in the dark for 30 min. After additional washing steps, macrophages were fixed for 15 min in 4% paraformaldehyde (PFA).

Techniques: Irradiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Scientific Reports

Article Title: Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

doi: 10.1038/srep18765

Figure Lengend Snippet: ( A) Actin (green) and tubulin (red) stainings of non-irradiated and 10 Gy irradiated macrophages ( n = 4). Quantification of cell area and aspect ratio was performed using Fiji software. Scale bar indicates 20 μm. ( B ) Phagocytic ability of non-irradiated and 10 Gy-irradiated macrophages ( n = 5) was determined after 1 h incubation with FITC-labelled (green) Staphylococcus aureus particles. F-actin was stained with rhodamine phalloidin (red). The percentage of macrophages able to phagocyte S.aureus particles was quantified using Fiji software. Scale bar indicates 20 μm. ( C ) MMP-2 and -9 activity was evaluated by gelatin zymography, using 1 and 15 μg of protein from CM of non-irradiated and 10 Gy-irradiated macrophages ( n = 10). White bands of proteolytic activity were revealed on a Coomassie Blue-stained gelatin gel. All data was analysed with paired t -test. ** P < 0.01, *** P < 0.001. Median is represented by the horizontal line inside the box plots, while the average is indicated by a “+”.

Article Snippet: Stainings with anti-human CD14-APC (clone MEM-18), HLA-DR-PE (MEM-12), CD86-FITC (clone BU63) (Immunotools) and CD163-PE (clone GHI/61) (R&D Systems) antibodies were performed in the dark for 30 min. After additional washing steps, macrophages were fixed for 15 min in 4% paraformaldehyde (PFA).

Techniques: Irradiation, Software, Incubation, Staining, Activity Assay, Zymography

Journal: Scientific Reports

Article Title: Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

doi: 10.1038/srep18765

Figure Lengend Snippet: Two main macrophage functional polarization status are recognized: a pro-inflammatory, responsible for killing intracellular pathogens and antitumour activity, and an anti-inflammatory one, which induces tissue repair, angiogenesis and promotes tumour activity. Pro-inflammatory macrophages produce high levels of TNF-α, IL-6 and IL-1β cytokines and exhibit CD80, CD86, HLA-DR, CCR7 and NF-κB increased expression, while anti-inflammatory ones express CD163, MRC1 and produce high levels of TGF-β1 and IL-10 cytokines. In the present study, we demonstrated that irradiated macrophages exhibit a decrease of anti-inflammatory ( CD163 , MRC1 and IL-10) and an increase of other pro-inflammatory ( CD80 , CD86, HLA-DR) markers. Although irradiated macrophages are more effective than non-irradiated ones at phagocytosis, a typical feature of pro-inflammatory macrophages, they fail to reach a classical pro-inflammatory phenotype, as they do not produce high levels of TNF-α, IL-6, IL-1β and CCR7. On the other hand, and similarly to their counterparts, irradiated macrophages are able to promote cancer cell invasion and cancer cell-induced angiogenesis. Our data suggests that M-CSF differentiated macrophages, exposed to cumulative ionizing radiation doses up to 10 Gy, exhibit a reduced anti-inflammatory-like phenotype, compared to non-irradiated ones, probably moving towards a pro-inflammatory phenotype. However, although irradiated macrophages exhibit characteristics from both pro- and anti-inflammatory phenotypes, they do not perfectly match to any of these typical profiles, appearing to acquire intermediate characteristics.

Article Snippet: Stainings with anti-human CD14-APC (clone MEM-18), HLA-DR-PE (MEM-12), CD86-FITC (clone BU63) (Immunotools) and CD163-PE (clone GHI/61) (R&D Systems) antibodies were performed in the dark for 30 min. After additional washing steps, macrophages were fixed for 15 min in 4% paraformaldehyde (PFA).

Techniques: Functional Assay, Activity Assay, Expressing, Irradiation