Visualizing Apoptosis with Annexin V

Cells grown on 35 mm glass-bottom MatTeK Petri dishes (MatTek Corporation, Ashland, MA, USA) were incubated for 20 min on ice with 5 µl Alexa Fluor 568-conjugated annexin V, washed twice with TBSS for 2 min, and subjected to fluorescence microscopy. To study annexin V binding to lipid membranes, giant vesicles were prepared as described previously (Weingärtner et al., 2012 (link)), diluted 1:2 in Ca²⁺-free or Ca²⁺-containing binding buffer supplemented with 1 µl Alexa Fluor 568-conjugated annexin V and incubated for 10 min before fluorescence microscopy.

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Grifell-Junyent M., Baum J.F., Välimets S., Herrmann A., Paulusma C.C., López-Marqués R.L, & Günther Pomorski T. (2021). CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts. Journal of Cell Science, 135(5), jcs258649.

Publication 2021

MatTeK Petri dishes

Alexa 568 Annexin v Buffer Cells Dishes Fluorescence microscopy Giant Membranes lipid

Corresponding Organization :

Other organizations : University of Copenhagen, Ruhr University Bochum, Humboldt-Universität zu Berlin, University of Amsterdam, Amsterdam University Medical Centers

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Variable analysis

independent variables

Presence or absence of Ca2+ in the binding buffer

dependent variables

Annexin V binding to lipid membranes measured by fluorescence microscopy

control variables

Cells grown on 35 mm glass-bottom MatTeK Petri dishes
Incubation time with Alexa Fluor 568-conjugated annexin V (20 min on ice)
Washing with TBSS (2 min, twice)
Preparation of giant vesicles as described previously (Weingärtner et al., 2012)
Dilution of giant vesicles in binding buffer (1:2)
Incubation time with Alexa Fluor 568-conjugated annexin V (10 min)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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