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A Mitotic HeLa Cell Elaborating Long Filopodia Revealed By a Membrane Marker (Grey), With the Entire Field Labeled For Mitochondria (Magenta)
by Naghmana Ashraf, New Mexico State University
Co-author: Charles Brad Shuster
Preparation: Cervical adenocarcinoma (HeLa) cells were cultured onto a glass-bottom MatTek dish in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2.5 mM L-Glutamine, 1.0 mM sodium pyruvate and 2.5 mM sodium bicarbonate, and maintained at 37ºC and 5% CO2. HeLa cells were transiently transfected with a membrane-tagged caspase biosensor (that was developed by inserting a caspase cleavage site between a myristoylation sequence and mApple), using Lipofectamine 2000 according to manufacturer’s instructions. After 6 h of transfection, cells were allowed to grow in fresh media overnight. The next day, cells were stained with mitochondrial potential sensitive Mitoview dye for 15 minutes. Cells were then subjected to live-cell imaging using an Andor Dragonfly 505 confocal imaging system.
A Mitotic HeLa Cell Elaborating Long Filopodia Revealed By a Membrane Marker (Grey), With the Entire Field Labeled For Mitochondria (Magenta)
by Naghmana Ashraf, New Mexico State University
Co-author: Charles Brad Shuster
Preparation: Cervical adenocarcinoma (HeLa) cells were cultured onto a glass-bottom MatTek dish in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2.5 mM L-Glutamine, 1.0 mM sodium pyruvate and 2.5 mM sodium bicarbonate, and maintained at 37ºC and 5% CO2. HeLa cells were transiently transfected with a membrane-tagged caspase biosensor (that was developed by inserting a caspase cleavage site between a myristoylation sequence and mApple), using Lipofectamine 2000 according to manufacturer’s instructions. After 6 h of transfection, cells were allowed to grow in fresh media overnight. The next day, cells were stained with mitochondrial potential sensitive Mitoview dye for 15 minutes. Cells were then subjected to live-cell imaging using an Andor Dragonfly 505 confocal imaging system.