Technical Reference #1914

1914.

PI3K-; actin- and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes Pirjo Spuul; Giuseppe Balistreri; Leevi Kääriäinen; Tero Ahola, University of Helsinki, Journal of Virology, 84(1914), (2010)
Link To Paper

Keywords:
Alphavirus; Replication Complex; Spherule; Membrane; Endocytosis

Materials & Methods:
For live cell imaging cells were grown in no. 0 glass-bottom culture dishes (MatTek Ashland MA) and MEM supplemented with 0.2% BSA 2 mM L-glutamine and 20 mM Hepes (pH 7.4) was used as imaging medium. Olympus IX-71 TILL imaging system supplemented with 37ºC chamber and CO2 was used to image the live cells with a UPlanSApo 60xW/120 water objective. Images were acquired with the rate of 2 frames/s (2 Hz). Leica TCS SP2 confocal microscope supplemented with 37ºC chamber and CO2 was used to image the live cells with a HCX PL APO 63x/12 W Corr/017 CS water objective. Live cell recordings were done with bidirectional scanning at 800 Hz speed; image stacks were acquired with the rate of 0.5 or 1 frame/s (0.5 or 1 Hz) as specified in figure legends. To visualize the acidic compartment of the cell lysotracker was added to the sample 20 min before imaging and it was present during the recordings.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
BHK-21 cells