Electron Microscopy Analysis of Cellular Organelles

For electron microscopy analysis, SKBR-3 and BT474 cells were seeded on glass chamber slides (Lab-Tek 177,380, Nalge Nunc int., Rochester, NY, USA) and treated for 7d. After drug treatments, cells were processed for electron microscopy [32 (link)] and observed with a CM10 electron microscope (Philips, Eindhoven, The Netherlands). Digital images were taken with a Megaview 3 camera. Analysis of morphologically identified multivesicular bodies (MVBs), autolysosomes (AL), autophagic vesicle/lipid droplets (AV/LDs) and lipid droplets (LDs) diameters were assessed in 10 cells for each treatment. The diameter of each organelle was measured with iTEM software package (Olympus-SYS; Olympus Corporation, Shinjuku, Tokyo, Japan) and plotted as histograms.

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D’Alesio C., Bellese G., Gagliani M.C., Aiello C., Grasselli E., Marcocci G., Bisio A., Tavella S., Daniele T., Cortese K, & Castagnola P. (2017). Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells. Journal of Experimental & Clinical Cancer Research : CR, 36, 154.

Publication 2017

CM10 electron microscope iTEM software package

Autolysosomes Autophagic Cells Drug Lipid droplets Microscope electron Multivesicular bodies Organelle

Corresponding Organization :

Other organizations : University of Genoa, Ospedale Policlinico San Martino, San Raffaele University of Rome

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Drug treatments

dependent variables

Diameters of multivesicular bodies (MVBs)
Diameters of autolysosomes (AL)
Diameters of autophagic vesicle/lipid droplets (AV/LDs)
Diameters of lipid droplets (LDs)

control variables

SKBR-3 and BT474 cells
Glass chamber slides (Lab-Tek 177,380, Nalge Nunc int., Rochester, NY, USA)
Incubation time of 7 days
CM10 electron microscope (Philips, Eindhoven, The Netherlands)
Megaview 3 camera for digital image capture
ITEM software package (Olympus-SYS; Olympus Corporation, Shinjuku, Tokyo, Japan) for diameter measurements

controls

No explicit mention of positive or negative controls

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