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Co-culture of BMDMs and MEFs in stiff 3D hydrogels increases CD54 and CD140a expression on macrophages. BMDMs and MEFs were co-cultured in 3D collagen-vitronectin, collagen-vitronectin-5% alginate scaffolds, or 2D controls for 24 hours. The viability, proportion of each cell type and surface expression of key markers was determined by flow cytometry. (A) Representative gating strategy for the separation and analysis of co-cultured BMDMs and MEFs by flow cytometry. Sample shown are BMDMs and MEFs cultured in stiff collagen-vitronectin-5% alginate hydrogels. (B) Overall cell viability. Note that this represents a mixture of BMDMs and MEFs. (C) Proportion of BMDMs (CD45 + CD11b + F4/80 + ) and MEFs (CD45 - CD11b - F4/80 - ) in the indicated co-culture conditions. (D, E) Geometric mean fluorescence intensity (gMFI) of CD11b and F4/80 expression on co-cultured BMDMs. (F) Representative histograms for CD54 and CD140a expression on BMDMs (left) and MEFs (right). (G–J) The relative expression of CD54 (G, H) and CD140a (I, J) on BMDMs and MEFs after co-culture (G, I) or single-culture (H–J) in the indicated models. Some single-cultured BMDMs were also activated with LPS and IFN-γ or IL-4 and IL-13, as indicated. Data in G-J are the relative gMFI, calculated by dividing gMFI values by the mean gMFI of unactivated BMDMs cultured in 2D. B-E and G-J show the pooled means of 3–4 independent experiments. Errors are SEM. Groups in B, D, E, H and J were tested by one-way ANOVA; C, G and I by two-way ANOVA where for the latter overall ANOVA results are indicated above each graph. Post-hoc analyses were performed for one- and two-way ANOVAs where significance between groups is indicated as **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

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Figure Lengend Snippet: Co-culture of BMDMs and MEFs in stiff 3D hydrogels increases CD54 and CD140a expression on macrophages. BMDMs and MEFs were co-cultured in 3D collagen-vitronectin, collagen-vitronectin-5% alginate scaffolds, or 2D controls for 24 hours. The viability, proportion of each cell type and surface expression of key markers was determined by flow cytometry. (A) Representative gating strategy for the separation and analysis of co-cultured BMDMs and MEFs by flow cytometry. Sample shown are BMDMs and MEFs cultured in stiff collagen-vitronectin-5% alginate hydrogels. (B) Overall cell viability. Note that this represents a mixture of BMDMs and MEFs. (C) Proportion of BMDMs (CD45 + CD11b + F4/80 + ) and MEFs (CD45 - CD11b - F4/80 - ) in the indicated co-culture conditions. (D, E) Geometric mean fluorescence intensity (gMFI) of CD11b and F4/80 expression on co-cultured BMDMs. (F) Representative histograms for CD54 and CD140a expression on BMDMs (left) and MEFs (right). (G–J) The relative expression of CD54 (G, H) and CD140a (I, J) on BMDMs and MEFs after co-culture (G, I) or single-culture (H–J) in the indicated models. Some single-cultured BMDMs were also activated with LPS and IFN-γ or IL-4 and IL-13, as indicated. Data in G-J are the relative gMFI, calculated by dividing gMFI values by the mean gMFI of unactivated BMDMs cultured in 2D. B-E and G-J show the pooled means of 3–4 independent experiments. Errors are SEM. Groups in B, D, E, H and J were tested by one-way ANOVA; C, G and I by two-way ANOVA where for the latter overall ANOVA results are indicated above each graph. Post-hoc analyses were performed for one- and two-way ANOVAs where significance between groups is indicated as **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

Article Snippet: Where indicated, BMDM monocultures were activated with 10 ng/mL lipopolysaccharide (LPS; tlrl-3pelps, Invivogen) and 10 ng/mL murine interferon gamma (IFNγ; Abcam), or 10 ng/mL murine interleukin (IL-4; PeproTech) and 10 ng/mL IL-13 (PeproTech), as indicated.

Techniques: Co-culture Assay, Expressing, Cell Culture, Flow Cytometry, Analysis, Fluorescence