Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 20

solution is prepared by mixing 5 mM PBFI-AM in DMSO with
10% Pluronic-127. The stock solution is diluted upon use to the
fi nal concentration using the medium (in millimoles): 120 NaCl,
25 Tris-HCl, 5.4 KCl, 1.8 CaCl 2 , and adjusted to pH 7.4. Cells
are incubated in 1.5 ml medium containing 5 M PBFI-AM for

40 min at 37°C. After loaded with PBFI-AM, cells are washed
twice with medium. PBFI-AM fl uorescence images in cell bodies
are acquired using an inverted microscope with a 40× objective
lens; the dye is excited at 340 nm. The dye can be sequentially
excited at 340 and 380 nm (510 nm emission). Pairs of images are
acquired at 5-7 s intervals which allows calculation of a ratio of
the fl uorescence images acquired at each excitation wavelength
(340/380 nm); thus data can be presented as the ratio for each
cell. The K + effl ux elicited by the specifi c K + ionophore valinomycin
should be used as a positive control. The images of PBFI-AM fl uo-
rescence can be analyzed offl ine using software such as Image J (NIH).
Several publications provide detailed information on this imaging
technique ( 42-45 ).
Caveats . Although PBRI has been widely used for measurement of
intracellular K+, it has fairly high affi nity to Na + as well (~1:10 for
Na + vs. K + ). Therefore, tight controlling for Na + infl ux and intrac-
ellular Na + concentration are key for an accurate measurement
of intracellular K + . Addition of TTX may be necessary in order to
block sodium channels during the measurement.

μ

The formation of pores in the mitochondrial membrane—the
mitochondrial membrane permeability transition (MPT)—is often
referred to as the “point of no return” in ischemia-triggered intrin-
sic apoptotic pathways. Disruption of mitochondrial membranes
not only allows the release of factors that can trigger or promote
the enzymatic degradation of the cellular components (cytochrome
c, smac/DAIBLO, AIF) but can lead to the loss of vital mitochon-
drial functions critical for maintaining cellular health and metabolism
[bioenergenic and biosynthetic activities (i.e., ATP production)],
and the production of excess reactive oxygen species (ROS).
Method . There are many useful histological and biochemical markers
for each of the several steps of the MPT. These include visualizing
the translocation of Cyt c, AIF and smac/DIABLO to the cytosol,
and the use of REDOX-sensitive dyes and indicators to monitor
the alterations/loss in mitochondrial membrane potential (

5.1.2. Middle Hallmarks

Mitochondrial Membrane
Permeability Transition

m )
Western blot analysis for any of these components usually entail
performing a (standard) subcellular fractionation to obtain mito-
chondrial and cytosolic preparations. Two recent, excellent sources
describing these techniques in detail are refs. ( 46, 47 ).
Caveats . Although a hallmark of the intrinsic apoptotic pathway,
translocation of cytochrome C to the cytoplasm is not by itself
dispositive for apoptotic cell death, it can occur in several other
pathways.

Δ Ψ