Figure 2

Functionality of bone marrow (BM)-derived macrophages after spraying. (a) Flow cytometric analysis of cell viability by propidium iodide (PI) staining of unsprayed and sprayed BM-derived macrophages (Left: representative images, right: quantification of n = 3, mean ± SEM). (b) Papenheim staining of cytospin preparations of sprayed and unsprayed BM-derived macrophages (scale bar: 20 µm) (c) Flow cytometric analysis of CD45.1, CD11b and F4/80 expression of unsprayed and sprayed BM-derived macrophages. Overlays of representative histograms; grey filled line: isotype, red/blue line: surface marker of unsprayed or sprayed macrophages. Lower panels: Delta mean fluorescent intensity (MFI) of surface marker expression of unsprayed and sprayed BM-derived macrophages (delta MFI was calculated by subtracting isotype (negative) MFI from surface marker (positive) MFI; n = 3, mean ± SEM). (d) Phagocytic activity of unsprayed and sprayed BM-derived macrophages. Upper panels: representative fluorescent microscopy images of BM-derived macrophages 2 hours after incubation with pHRodo E. coli BioParticles (scale bar: 200 µm). Lower panel: Representative flow cytometric analysis (grey filled line: cells without E. coli BioParticles, red line: unsprayed cells with E. coli BioParticles, blue line: sprayed cells with E. coli BioParticles) and quantification of n = 3 (mean ± SEM). (e) Analysis of MHC-II surface marker expression by flow cytometry of unsprayed and sprayed BM-derived macrophages before and after stimulation with IFNγ. Left: Representative histograms (grey filled line: isotype, green line: non-stimulated control, blue line: IFNγ stimulated) and right: Quantification of the fold change in MFI, n = 3, mean ± SEM). (n.s: denotes not significant. Statistical analysis: a: two-way repeated measurements (RM) ANOVA with Bonferroni post-hoc-test; (c–e) two-tailed paired student t-test).