Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Integrin-mediated phagocytic uptake is modulated by substrate stiffness hMDMs were seeded on fibronectin-coated polyacrylamide hydrogels or glass coverslips and incubated at 37°C for 4 h. Cells were pre-treated or not with 150 ng/mL PMA for 15 min, challenged with opsonized RBCs (iC3b-IgM-RBCs) and fixed immediately (time “0” min) or after 15 min at 37°C. Cells were then labeled with Alexa Fluor 488-conjugated phalloidin and a Cy3-labeled anti-rabbit antibodies that recognize the anti-sheep RBCs IgMs used to opsonize RBCs. (A) Representative wide-field images of macrophages seeded on 4 kPa, 12 kPa, or 25 kPa hydrogels or glass and incubated with iC3b-IgM-RBCs for 15 min. Opsonized RBCs are shown in cyan in the upper panel and phase contrast images are shown in the lower panel, where phagosomes are visible (examples of phagosomes are shown by the white arrows). Scale bar, 10 μm. (B and C) Quantification of RBC association at 0 min (B) and phagocytosis after 15 min (C) on the different substrates, with or without PMA pre-treatment. Association refers to both bound and internalized RBCs while phagocytosis only considers internalized RBCs. (D) Phagocytosis efficiency after 15 min on the different substrates, with or without PMA pre-treatment, calculated as the percentage of bound RBCs that are internalized. (E) Representative confocal images of macrophages seeded on 4 kPa, 12 kPa, or 25 kPa hydrogels or glass and incubated with iC3b-IgM-RBCs for 2 min. F-actin is shown in gray while RBCs are shown in cyan (white arrow shows one example of an actin cup). Scale bar, 10 μm. (F) Quantification of the number of actin cups formed by macrophages after 2 min. (B–D and F) Data from each donor is color coded: individual cells are represented as the smaller, lighter dots; means of all cells for each donor are shown as the bigger, darker dots with a black border. 25–30 cells were analyzed per condition and per donor. Horizontal bars represent the means of all donors. Ratio paired t tests were used to compare the means of all donors. ∗: p value < 0.05; ∗∗: p value < 0.01.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Incubation, Labeling

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Analysis of the arrangement of traction forces exerted by cells on their substrate highlights differences between control and phagocytosing macrophages hMDMs were seeded on fibronectin-coated 10 kPa polyacrylamide gels and incubated at 37°C for 4 h. Cells were challenged or not with opsonized RBCs (iC3b-IgM-RBCs) and imaged live for 30 min at 37°C. (A and B) (Upper panel) Traction force distribution of a control cell (A) and a phagocytosing cell (B) at different time points; also see and . (Lower panel) Corresponding brightfield images with traction force vectors superimposed in red for the control cell (A) and the phagocytosing cell (B). Green circles represent the approximate outlines of the cells. One example of an RBC about to be internalized is depicted by the white arrow. Time is shown as minutes:seconds. Scale bar, 10 μm. (C) Polar degree of the control cell (red) and the phagocytosing cell (blue) over the 30 min observation period. The orange line (polar degree equal to 1) corresponds to a theoretical situation with a purely uniaxial arrangement of traction forces and the green line (polar degree equal to 0) represents a purely isotropic arrangement. (D) Polar degree of 8 control cells (red) and 7 phagocytosing cells (blue) from independent donors at 0 and 30 min. Unpaired t tests were used to compare the populations of control and phagocytosing cells at each time point. ∗: p value < 0.05.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Control, Incubation

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Phagocytosing macrophages show greater changes in their main traction than control cells hMDMs were seeded on fibronectin-coated 10 kPa polyacrylamide gels and incubated at 37°C for 4 h. Cells were challenged or not with opsonized RBCs (iC3b-IgM-RBCs) and imaged live for 30 min at 37°C. (A and B) (Upper panel) Traction force distribution of a control cell (A) and a phagocytosing cell (B) at different time points; also see and . (Lower panel) Corresponding brightfield images with main traction superimposed in orange for the control cell (A) and the phagocytosing cell (B). Green circles represent the approximate outlines of the cells. Time is shown as minutes:seconds. Scale bar, 10 μm. (C) Change in main traction (in degrees) for the control cell (red) and the phagocytosing cell (blue) over the 30 min observation period. (D) Change in main traction (in degrees) for 11 control cells (red) and 13 phagocytosing cells (blue) from independent donors observed for 30 min. Each column corresponds to a cell and each dot represents one change in main traction. (E) Standard deviation of change in main traction for 11 control cells (red) and 13 phagocytosing cells (blue) over the 30 min observation period. A Mann-Whitney test was used to compare the populations of control and phagocytosing cells. ∗: p value < 0.05.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Control, Incubation, Standard Deviation, MANN-WHITNEY

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Phagocytosis is associated with the disruption of podosomes at the ventral side of macrophages hMDMs were seeded on glass coverslips, incubated at 37°C for 4 h and challenged with opsonized RBCs (iC3b-IgM-RBCs). Cells were fixed at steady state (basal) or after a 2- or 15-min incubation with RBCs, permeabilized and labeled with Alexa Fluor 488-conjugated phalloidin and Cy3-labeled anti-rabbit antibodies that recognize the anti-sheep RBCs IgMs used to opsonize RBCs. (A) Representative confocal images of the ventral side of macrophages (close to the coverslip) at steady state (basal) or after 2 or 15 min with RBCs. F-actin is shown in gray while RBCs are shown in cyan. Scale bar, 10 μm. (B and C) Quantification of podosome density (B) and cell spreading area (C) at the different time points. Each dot represents an individual cell; data are pooled from 3 independent donors with 25–30 cells per condition and per donor. Because podosome density at steady state across the different donors is heterogenous, results are expressed as fold change from the mean of all cells in the basal condition for each donor. A Kruskal-Wallis test was used for comparison of multiple groups in (B). (D) Representative confocal images of the ventral side (upper panel) or dorsal side (lower panel) of macrophages after 2 min with RBCs. F-actin is shown in gray while RBCs are shown in cyan. Blue dashed box highlights a macrophage with F-actin recruitment around multiple RBCs on the dorsal side (white arrow shows one example of an actin cup) and no podosome on the ventral side; orange dashed box shows a macrophage with no F-actin recruitment around bound RBCs on the dorsal side and many podosomes on the ventral side. Scale bar, 10 μm. (E) Quantification of podosome density after 2 min with RBCs, comparing macrophages with F-actin recruitment around RBCs (actin cups) or not. Each dot represents an individual cell; data are pooled from 3 independent donors with 25–30 cells per donor. A Mann-Whitney test was used to compare the two cell populations. ∗: p value < 0.05; ∗∗∗∗: p value < 0.0001.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Disruption, Incubation, Labeling, Comparison, MANN-WHITNEY

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Relocalization of proteins associated with podosomes or phagocytic cups during integrin-mediated phagocytosis hMDMs were seeded on glass coverslips, incubated at 37°C for 4 h and challenged with opsonized RBCs. Cells were fixed at steady state (basal) or after a 2-min incubation with RBCs, permeabilized and labeled with Alexa Fluor 488-conjugated phalloidin and a mouse antibody that recognizes vinculin (A), rabbit antibodies against p34-Arc/ARPC2 (B) or a recombinant antibody that recognizes RhoA-GTP (C), followed by Alexa Fluor 647-labeled anti-mouse, anti-rabbit or anti-human IgG antibodies. Representative confocal images of the ventral side (upper panel) or dorsal side (lower panel) of macrophages at steady state (left) or during phagocytosis of opsonized RBCs (right). On merge images, F-actin is shown in red and vinculin, ARPC2 or RhoA-GTP is shown in green. Scale bar, 10 μm.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Incubation, Labeling, Recombinant

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet: Integrin-mediated phagocytosis decreases gelatin degradation by macrophages hMDMs were seeded on fluorescent gelatin-coated coverslips and incubated at 37°C for 1 h and challenged with opsonized RBCs (iC3b-IgM-RBCs). Cells were fixed after 2 h, permeabilized and labeled with Alexa Fluor 633-conjugated phalloidin and Cy3-labeled anti-rabbit antibodies that recognize the anti-sheep RBCs IgMs used to opsonize RBCs. (A) Representative wide-field images of macrophages challenged (lower panel) or not (upper panel) with RBCs. F-actin is shown in gray, RBCs in cyan and gelatin in green. Scale bar, 10 μm. (B) Quantification of mean degradation area per cell. Each dot corresponds to the mean of at least 30 cells per condition and per donor. A ratio paired t test was used to compare the means of all 4 donors. ∗: p value < 0.05.

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Incubation, Labeling

Journal: iScience

Article Title: A crosstalk between adhesion and phagocytosis integrates macrophage functions into their microenvironment

doi: 10.1016/j.isci.2025.112067

Figure Lengend Snippet:

Article Snippet: Anti-sheep red blood cells (RBCs) rabbit IgMs , Gentaur , Cat#MBS524107.

Techniques: Recombinant, Electron Microscopy, Software, Cell Culture

Journal: Scientific Reports

Article Title: Evidence, detailed characterization and clinical context of complement activation in acute multisystem inflammatory syndrome in children

doi: 10.1038/s41598-022-23806-5

Figure Lengend Snippet: Key resources.

Article Snippet: Anti-sheep red blood cell stroma antibody (rabbit) , Sigma-Aldrich , Cat# S1389.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Software