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Cells for real-time microscopy were plated onto glass bottomed tissue culture dishes (MatTek Corp).
Cultures containing granular starch-degrading microorganisms, as judged by the degradation of starch granules observed with light microscopy, were plated on medium containing 18 mM KH2PO4, 40 mM K2HPO4, 0.8 mM MgSO4 7H2O, 6.7 mM KCl, 0.07 mM FeCl3 6H23.523.5 mM NaNO3 and 2% (w/ v) agar, pH 7.2.
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To prepare for microscopy, cells were plated at ∼5×105 cells per coverslip (25 mm circular, No. 1.5) and transfected with plasmids using Roche FuGENE 6 according to the manufacturer's protocol (Roche Diagnostics).
For confocal microscopy cells were plated on similarly treated glass cover slips.
For fluorescence microscopy, cells were plated on glass coverslips at 80% confluence.
For immunofluorescence microscopy, cells were plated into glass-bottom dishes (MatTek).
For visualization by fluorescent microscopy, cells were plated in glass bottom dishes and treated with 0.3 mM H2O for 6 h.
For all kinds of living cell microscopy, cells were plated on 32 mm glass coverslips in a special chamber (Jacques Monod Institute, http://www.ijm.fr/en/ijm/facilities/imagoseine) with 2 ml of culture medium.
For live cell imaging using spinning disk confocal microscopy, cells were plated in 35 mm dishes with a 14 mm No. 1.5 thickness coverglass window in the bottom (MatTek Corporation, Ashland, MA).
For TIRF microscopy experiments, cells were plated for 2 h on glass-bottom dishes (MatTek Corporation) precoated with 10 μg ml−1 of bovine plasma FN overnight at 4 °C.
For time-lapse microscopy, T cells were plated on ICAM-1 or on confluent HUVECs grown on fibronectin-coated coverslips.
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