Journal:

Article Title: Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells * S⃞

doi: 10.1074/jbc.M804392200

Figure Lengend Snippet: S1P-mediated endothelial functions are impaired in senescent ECs. A, proliferative capabilities of young (Y, CPDL = 15), intermediate (I, CPDL = 35), and senescent (S, CPDL = 60) ECs. Cells were labeled with BrdUrd reagent for 24 h. Subsequently, cells were immunostained with anti-BrdUrd following the manufacturer's instructions (BrdUrd staining kit, Zymed Laboratories Inc.). Mean ± S.E. represents percentage of BrdUrd incorporated nuclei from 3-5 microscopic fields of duplicate experiments. B, chemotactic responses were measured by a Transwell migration assay as described under “Experimental Procedures.” Note that S1P (100 nm)-induced chemotactic responses were inversely related to in vitro ages of cultured ECs. C, young, intermediate, and senescent ECs were plated into each well of 8W1E arrays and incubated at 37 °C to allow the transendothelial electrical resistance (TEER) to reach equilibrium. 24 h later, the cells attached to the microelectrodes were killed by applying 5 V of electrical current for 30 s (arrow), which resulted in an abrupt impedance drop. Subsequently, endothelial wound-healing responses in the presence or absence of 100 nm S1P were determined in real-time by measuring the recovery of TEER, an indicator of endothelial migration into the wounded area (29). D, morphogenesis of young and senescent ECs on matrigel (upper panel) and quantitative analysis of tubular length (lower panel) in the presence or absence of S1P (200 nm) were performed as described (12, 13). Scale bar, 318 μm.

Article Snippet: Briefly, 200 μl of cell suspension (5 × 10 5 cells per ml) was seeded to each well of either an 8W1E or 8W10E ECIS array (Applied BioPhysics), which was pre-equilibrated with 200 μl of medium at 37 °C for 30 min. Two days later, endothelial cells were washed and replaced with plain M199 medium supplemented with 0.5% fetal bovine serum.

Techniques: Labeling, Staining, Transwell Migration Assay, In Vitro, Cell Culture, Incubation, Migration