Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange.

Abstract:

:Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Ferlini C,Scambia G

doi

10.1038/nprot.2007.397

subject

Has Abstract

pub_date

2007-01-01 00:00:00

pages

3111-4

issue

12

eissn

1754-2189

issn

1750-2799

pii

nprot.2007.397

journal_volume

2

pub_type

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