Senescent cells reduce the uptake of apoptotic corpses by macrophages. (A) Schematic overview of the experimental design using direct co-cultures between proliferating or senescent fibroblasts (normal healthy or IPF-derived) and primary macrophages. Cells were co-cultured for 16 h, and then exposed to Cell tracker green and CypHer5E co-labeled apoptotic corpses (Raji cells; 2.5:1 ratio). Co-cultures were washed 1, 2 or 4 h post feeding and subsequently fixed to perform immunofluorescence staining. (B) Immunofluorescence microscopy of MDM in indicated co-cultures (either proliferating or senescent primary IPF-derived fibroblasts) exposed to apoptotic Rajis (labeled with Cell tracker green [green] and the pH-sensitive dye CypHer5E [white]) for 1, 2 or 4 h were then washed, fixed, and stained for CD45 (red) and Hoechst (blue). Scale bar: 20 µm. Representative images are from two independent experiments. (C and D) Quantification of apoptotic corpse uptake by macrophages in co-culture with human MDMs and senescent or proliferating primary IPF-derived (C) or normal healthy fibroblasts (D). Measurements and analysis of the number of green events per image (cell tracker green labeled Raji cells) were performed using the IncuCyte S3 system. All values are means ± SEM. Statistically significant differences were determined by two-way ANOVA with Tukey correction; n = 6 biological replicates. *P < 0.05, **P < 0.001. (E and F) Quantification of the ratio between red (CypHer5E)⁺ green (Cell tracker)⁺ (overlap) events and the total number of green events in the same field of view. Data are obtained from co-cultures between MDMs and proliferating (black) or senescent (red) IPF-derived (E) or normal healthy fibroblasts (F) exposed to apoptotic Rajis as shown in A. All values are means ± SEM. n = 6 biological replicates.