Technical Reference #1944

1944.

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair Christina Tam; Vincent Idone; Cecilia Devlin; Maria Cecilia Fernandes; Andrew Flannery; Xingxuan He; Edward Schuchman; Ira Tabas; and Norma W. Andrews, Yale University School of Medicine, Journal of Cell Biology, 189(1944), (2010)
Link To Paper

Materials & Methods:
Live time-lapse imaging of FM1-43 influx Subconfluent cells plated on glass-bottom dishes (MatTek) were preincubated with SLO for 5 min at 4°C transferred to a LiveCell System chamber (Pathology Devices) at 37°C with 5% CO2 and exposed to prewarmed DME containing or not Ca2+ and 4 μM FM1-43 (Invitrogen) and SLO (Idone et al. 2008b). Spinning disk confocal images were acquired for 4 min at 1 frame/3 s using the UltraVIEW VoX system (PerkinElmer) attached to an inverted microscope (Eclipse Ti; Nikon) with a 40× NA 1.3 objective (Nikon) and equipped with a camera (C9100-50; Hamamatsu Photonics). Quantitative analysis of intracellular fluorescence was performed using Volocity Suite (PerkinElmer).

Microscopic Technique
Confocal Microscopy, Inverted Microscopy

Cell Type(s)
Subconfluent cells