changes in protein profiles in bortezomib applied multiple myeloma ...

3.2.6.3. Detection of the Loss of Mitochondrial Membrane Potential
(MMP)
Mitochondrion is responsible for mediating the release of cytochrome-c
molecules. Thus, it has a vital function in the induction of intrinsic apoptosis. Loss of
mitochondrial membrane potential (MMP), that is a hallmark for apoptosis, causes to
form pores and these pores allows to release of cytochrome-c into the cytoplasm. This
event known as the beginning of the apoptotic response. The decrease of MMP in MM
U-266 cells was assessed by APO LOGIX JC-1 MMP Detection Kit (Cell Technology,
USA). JC-1, which is a signal molecule to detected the changes in MMP, is a unique
cationic dye. In non-apoptotic cells, JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-
tetraethylbenzimidazolylcarbocyanine iodide) exist as a monomer in the cytosol (green)
and also accumulates as aggregates in the mitochondria which stain red. Whereas, in
apoptotic cells or unhealthy cells, JC-1 exist in monomeric form and stains the cytosol
to green. For this reason, apoptotic cells, showing only green fluorescence, are easily
differentiated from healthy cells that show intense red fluorescence. This is the basic
principle of the method.
Shortly, 1x10 6 cells were seeded in a 6-well plate in 2 ml growth medium in the
absence or presence of increasing concentrations of Bortezomib (1 nM, 10 nM, 20 nM)
at 37°C in 5% CO2 for 72 hours. Untreated cells were used as control group.
C
20 nM
1 nM
10 nM
Figure 3.5. Aplied Bortezomib Doses on U-266 Cells for Dedection of Loss of MMP
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