Fig. 1.

Validation of senescence green probe (SGP) as a marker of senescence. Ai-vi shows a representative gating strategy used to identify senescent cells populations in H₂O₂-treated cells. Focused cells were gated into single cell population (green box). DAPI⁺ cells (magenta box) was analyzed for p16 expression followed by SGP expression against cell area/size. Small p16⁺ cells (yellow box) shows a population with high SGP expression (intermediate senescent cells) and another population negative for SGP expression (non-senescent cells). A majority of the large cells with high expression of p16 (orange box) also had high expression of SGP (committed senescent cells). Untreated, H₂O₂-treated, and unstained HUVECs were analyzed by Amnis ImageStream and gated in the same fashion. B Representative images of unstained HUVECs, non-senescent HUVECs, and HUVECs at different stages of senescence according to their cell size in brightfield (BF) and side scatter (SSC) as well as expression of senescent markers SGP (green) and p16 (red). Committed senescent cell in the representative image shown here has two DAPI (purple) positive nucleus. C Quantification of the percentage of SGP⁺ and p16⁺ large senescent cells in untreated and H₂O₂-treated HUVECs. D Representative images of HUVECs at different stages of senescence according to cell size and senescent markers SGP (green) and p21 (red). E Quantification of the percentage of SGP⁺ and p21⁺ large senescent cells in untreated and H₂O₂-treated HUVECs. In vitro data presented for p16- or p21-independent experiments are from 2 different HUVEC lines and are presented as mean ± standard deviation, scale bar = 20 µm. F Representative images of traditional SA-β-gal (blue) and SGP (green) stain in the brain taken from APP/PS1 mice after amyloid plaque development. Amyloid beta (gray) staining shows that SGP (green) staining on CD31⁺ vasculature (red) is localized to vascular cells, not plaques deposited on the blood vessel. Scale bar = 50 µm