complement inhibitor compstatin  (Tocris)

Journal: iScience

Article Title: Polystyrene microplastics cross the murine intestine and induce inflammatory cell death after phagocytosis by human monocytes and neutrophils

doi: 10.1016/j.isci.2025.114076

Figure Lengend Snippet: Phagocytosis of 10 μm PS particles is complement-dependent (A) Phagocytosis of 10 μm Nile Red stained PS particles (red) by f-MLF stimulated calceinAM stained neutrophils (green) measured on a Leica Stellaris5 confocal microscope. PS particles were pre-incubated with human pooled serum (HPS). The right graph represents the fluorescent intensity of the green neutrophils (green graph line) and red 10 μm PS particles (white graph line) along the vertical green line in the image on the left. Timepoint shown is 120 min. (B) Phagocytosis of 10 μm Nile Red stained PS particles (red) by f-MLF stimulated calceinAM stained neutrophils (green) on a Leica Stellaris5 confocal microscope. PS particles were pre-incubated with HPS. Images on the right and bottom show the z dimension along the green lines in the XY image on the top left. Timepoint shown is 120 min. (C) Phagocytosis of 10 μm uncolored PS particles (gray) by f-MLF stimulated calceinAM stained neutrophils (green) on a Leica Stellaris5 confocal microscope. PS particles were pre-incubated with HPS or heat-inactivated HPS and subsequently cultured in HPS, HPS +28 μM compstatin, HPS +0.1 mg/mL FcR inhibitor, or heat-inactivated HPS. Timepoint shown is 120 min. Scale bar is 50 μm. Neutrophil phagocytosis at 120 min was manually quantified in two images per 3 different donors. Results are shown as means plus standard deviations. Controls were compared to the different treatments with a paired one-way ANOVA with Dunnett multiple comparison correction. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: In order to test the requirements of neutrophil phagocytosis of 10 μm PS particles, PS particles were pre-incubated with HPS or heat-inactivated HPS and subsequently cultured in HPS, HPS + 28μM of the complement inhibitor compstatin (Tocris bioscience), HPS + 0.1mg/ml of polyclonal antibodies binding and inhibiting the FcR (eBioscience) or heat-inactivated HPS.

Techniques: Staining, Microscopy, Incubation, Cell Culture, Comparison

Journal: Microorganisms

Article Title: C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

doi: 10.3390/microorganisms8071039

Figure Lengend Snippet: The interaction between polymorphonuclear neutrophils (PMNs) and Bacillus anthracis (hk Ba ) or anthrax peptidoglycan (PGN) is dependent on the complement opsonization of pathogen particles. ( A ) Flow cytometry gating strategy and exemplification of neutrophil purification (CD16 high leukocytes). ( B ) Histogram overlay of FITC-labelled hk Ba (left) and/or PGN (right) interaction with CD16 high PMNs isolated from a median responsive individual, in the presence of compstatin, a C3 convertase inhibitor, or the immunodepletion of complement factors.

Article Snippet: The C3 convertase complement inhibitor compstatin was from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Purification, Isolation, Immunodepletion

Journal: Microorganisms

Article Title: C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

doi: 10.3390/microorganisms8071039

Figure Lengend Snippet: Flow cytometry quantitation of FITC-hk Ba ( A ) and/or the FITC-PGN ( B ) interaction with CD16 high neutrophils in the presence of compstatin, a C3 convertase inhibitor, or the immunodepletion of complement factors. Data, shown as mean ± SD of 4–8 independent donors, depict bacteria or PGN positive PMNs (left panels), total bacteria or PGN uptake (geometric mean of fluorescence intensity, middle panels) and normalized changes in fluorescence intensity compared to bacteria or PGN uptake in the presence of autologous serum (AHS) that is considered 100% (right panels). For each panel, statistically significant differences compared to PAMPs + AHS (shaded) are depicted graphically (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) and were computed either by repeated measures one-way analysis of variance (ANOVA) with Holm–Sidak’s multiple comparisons test (left and middle panels), or by one sample t test compared to the normalized value (right panel).

Article Snippet: The C3 convertase complement inhibitor compstatin was from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Quantitation Assay, Immunodepletion, Bacteria, Fluorescence

Journal: Microorganisms

Article Title: C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

doi: 10.3390/microorganisms8071039

Figure Lengend Snippet: Quantitation of pHrodo-hk Ba ( A ) and/or pHrodo-PGN ( B ) internalization by neutrophils in the presence of compstatin, a C3 convertase inhibitor, or the immunodepletion of complement factors. Data are shown as mean ± SD of 6 independent donors, and depict pHrodo fluorescence intensity after the internalization of labeled bacteria or PGN (left panels) and normalized changes in endocytosed pHrodo-labeled particles compared to bacteria or PGN uptake in the presence of autologous serum (AHS), which was considered 100% (right panels). Statistically significant differences compared to pHodo uptake in the autologous serum (AHS, shaded) are depicted graphically (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001), and were computed by repeated measures one-way ANOVA with Holm–Sidak’s multiple comparisons test (left panels), or one sample t test compared to the normalized value (right panels).

Article Snippet: The C3 convertase complement inhibitor compstatin was from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Quantitation Assay, Immunodepletion, Fluorescence, Labeling, Bacteria

Journal: Microorganisms

Article Title: C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

doi: 10.3390/microorganisms8071039

Figure Lengend Snippet: Quantitation of neutrophil degranulation and myeloperoxidase (MPO) release after bacteria (hk Ba , A ) or PGN ( B ) stimulation in the presence of compstatin, a C3 convertase inhibitor, or the immunodepletion of complement factors. Data are shown as mean ± SD of 10 independent donors and depict MPO activity in supernatants collected from stimulated neutrophils (left panels), changes in released MPO after the normalization to bacteria or PGN stimulation in the presence of autologous serum (AHS), which was considered 100% (right panels). Unless otherwise marked, statistically significant differences compared to bacteria and/or PGN stimulation in the presence of AHS (shaded) are depicted graphically (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001), and were computed by repeated measures one-way ANOVA with Holm–Sidak’s multiple comparisons test (left panels) or one sample t test compared to the normalized value (right panel).

Article Snippet: The C3 convertase complement inhibitor compstatin was from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Quantitation Assay, Bacteria, Immunodepletion, Activity Assay

Journal: Microorganisms

Article Title: C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

doi: 10.3390/microorganisms8071039

Figure Lengend Snippet: Flow cytometry quantitation of myeloperoxidase (MPO) immunoreactivity in neutrophils stimulated with hk Ba ( A ) or PGN ( B ) in the presence of compstatin, a C3 convertase inhibitor, or the immunodepletion of complement factors. Data are shown as mean ± SD of 10 independent donors and the depict intracellular MPO fluorescence (geometric mean) in CD16 high neutrophils (left) or the changes in MPO fluorescence after paired normalization to control MPO immunoreactivity in the absence of agonists (right panel). Statistically significant differences compared to bacteria and/or PGN-induced MPO immunoreactivity in the presence of the autologous serum (AHS, shaded) are depicted graphically (* p < 0.05; ** p < 0.01; *** p < 0.001), and were computed by repeated measures one-way ANOVA with Holm–Sidak’s multiple comparisons tests.

Article Snippet: The C3 convertase complement inhibitor compstatin was from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Quantitation Assay, Immunodepletion, Fluorescence, Control, Bacteria