Tracking Exocytosis in COS-1 Cells

COS-1 cells were plated at low density on glass coverslips and transfected with APP-SEP (super ecliptic pHluorin) and with pARIS HTT or pARIS HTT_SA (Pardo et al., 2010 (link)) using calcium phosphate. Acquisitions were made the day after transfection at 5 Hz during 1 min using an inverted microscope (Elipse Ti, Nikon) with a X60 1.42 NA APO TIRF oil-immersion objective (Nikon) coupled to a CCD camera (CoolSnap, Photometrics) and maintained at 37°C and 5% CO2. Analysis was done on area delimited by cell edges and exocytosis rate was quantified using ExocytosisAnalyser macro on ImageJ developed by Marine Scoazec.

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Bruyère J., Abada Y.S., Vitet H., Fontaine G., Deloulme J.C., Cès A., Denarier E., Pernet-Gallay K., Andrieux A., Humbert S., Potier M.C., Delatour B, & Saudou F. (2020). Presynaptic APP levels and synaptic homeostasis are regulated by Akt phosphorylation of huntingtin. eLife, 9, e56371.

Publication 2020

Elipse Ti X60 1.42 NA APO TIRF oil-immersion objective

Calcium phosphate Cell Cos 1 cells Exocytosis Immersion Marine Microscope Phluorin Transfection

Corresponding Organization :

Other organizations : CEA Grenoble, Inserm, Université Grenoble Alpes, Centre Hospitalier Universitaire de Grenoble, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Grenoble Institute of Neurosciences, Centre National de la Recherche Scientifique, Sorbonne Université, Institut du Cerveau

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Variable analysis

independent variables

Transfection with APP-SEP (super ecliptic pHluorin)
Transfection with pARIS HTT or pARIS HTT_SA

dependent variables

Exocytosis rate

control variables

COS-1 cells plated at low density on glass coverslips
Maintained at 37°C and 5% CO2
Acquisition at 5 Hz during 1 min using an inverted microscope (Elipse Ti, Nikon) with a X60 1.42 NA APO TIRF oil-immersion objective (Nikon) coupled to a CCD camera (CoolSnap, Photometrics)

controls

Positive control: Not specified
Negative control: Not specified

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