Fig. 1: Analysis of senescence markers in long-term cultures of human monocyte-derived macrophages and schematic representation of the experimental model. | Communications Biology

Fig. 1: Analysis of senescence markers in long-term cultures of human monocyte-derived macrophages and schematic representation of the experimental model.

From: Immunoproteasome remodeling in senescing human macrophages reveals the loss of PA28αβ capping as a hallmark of immunosenescence

Fig. 1

a Optical microscope images of cells left unstained (Bright field) and stained with Sudan B black or for (SA)β-gal activity. Red arrows indicate lipofuscin-positive cells. Scale bars 30 µm. Cells were analyzed after 3, 7, 14 and 28 days of culture. b Five micrograms of total protein (for the detection of p21Waf1/Cip1) and (c) 10 µg of total protein (for the detection of p16INK4a) were separated via SDS‒PAGE on a 14% (w/v) acrylamide gel and subjected to western immunoblotting with specific antibodies. The total protein content in each lane was detected using No Stain reagent as a loading control. Immunoreactive bands and total protein signals were imaged with a ChemiDoc system and quantified with Image Lab software. The values were normalized to the total protein content and are reported in the graph as the fold change vs. day 7. Data are the mean ± S.D. *p < 0.05; **p < 0.01; ***p < 0.005. d Coexpression of the p21Waf1/Cip1 and p16INK4a proteins by immunofluorescence confocal microscopy at 14 days. Nuclei were stained with DRAQ5 reagent, and images were captured using a confocal Leica microscope (63× oil objective). Scale bar 15 µm (e) Phagocytosis/lysosomal activity was assessed by flow cytometry using a pHrodo Green Zymosan BioParticles Conjugate assay kit. MFI signals were normalized to the lysosome content, as determined by LysoTracker Red (LTR) staining. Data are the mean ± S.D. (*p < 0.05). f After PMBC isolation, monocytes (M0) were separated from lymphocytes by plastic adhesion and cultured for up to 28 days; after 7 days of culture, adherent MOs (yellow) differentiated into activated macrophages (Mфs, red); if kept in culture for up to 3 weeks, Mфs were deactivated over time (Mф, green), and a senescence-like phenotype developed (S-Mф, gray). On the basis of senescent marker expression, day 14 coincided with the onset of senescence, and day 28 coincided with the senescent state.

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