Research Paper Volume 8, Issue 7 pp 1294—1315

Figure 1. SC implantation in vivo. (A-B) Bioluminescent signal accumulation in a cohort (n=5 mice) of chronologically aged mice harboring a hemizygous p16(Ink4a) knock-in of luciferase (p16^LUC mice; p16^Ink4a/Luc). (A) Whole body luminescence (total flux; p/s) for individual mice are depicted. (B) Serial bioluminescence imaging of chronologically aged mice. Color scale indicates signal intensity (same thresholds across all time points). (C-D) A model of SC implantation into SCID mice. NDF cells harboring a secreted GLuc reporter construct (NDF-GLuc) were implanted intraperitoneally into SCID mice as microcarrier bead cultures that were, prior to injection, cultured in low serum (0.2% FBS) for induction of quiescence (Qui NDF) or irradiated at 20 Gy for induction of senescence (Sen NDF). Alternatively, irradiated NDFs were coated in protective alginate gel (Sen NDF + Alg). Kinetics of NDF-GLuc survival was monitored via measurement of GLuc activity in mouse plasma collected at regular intervals over 28 days. The amount of GLuc activity remaining in the blood over time is expressed as a percentage of activity in plasma 24 hours after cell inoculation. Values depicted are means ± SEM for each group (n = 4-6 mice/group). Differences between all groups are statistically significant after day 7 (p≤0.001). (D) Microphotographs of empty alginate beads (no cells) and alginate beads containing embedded irradiation-induced senescent NDFs (bright field images). After embedding senescent NDF cells and before implantation into mice, viability of embedded cells was assessed by labeling live cells with Calcein AM (green) and dead cells with propidium iodide (PI; red), followed by fluorescent microscopy. Senescent NDFs in alginate beads were also assessed for β-gal^pH6 staining. Representative images are shown (magnification 100x). Successful embedding of cells was indicated by >90% viability (< 10% PI-positive cells).