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

In Figure 3.5 ;
C is correspond to control group (1x10 6 U-266 Cells / 2 ml / well),
1 nM is correspond to 1 nM Bortezomib concentration (1x10 6
U-266 Cells / 2 ml / well + 1 nM Bortezomib in 2 ml),
10 nM is correspond to 10 nM Bortezomib concentration (1x10 6
U-266 Cells / 2 ml / well + 10 nM Bortezomib in 2 ml),
20 nM is correspond to 20 nM Bortezomib concentration (1x10 6
U-266 Cells / 2 ml / well + 20 nM Bortezomib in 2 ml),
After this period, all samples (control group and the cells induced to undergo
apoptosis) are taken to the falcon tubes respectively and washed with 1X PBS before
the centrifugation at 1000 rpm for 10 minutes. Supernatants were carefully removed
from the pellet and discarded. Then the pellets were dissolved in 500 μl of JC-1 dye and
the cells were incubated at 37˚C in 5% CO2 for 15-30 minutes. Next, the mixtures were
centrifuged at 400g (~1000 rpm) for 5 minutes and supernatants were carefully removed
again. Subsequently, 2 ml of 1X Assay Buffer was added onto the pellets and vortexed
till to be sure that it was homojenized. Immediately afterwards centrifugation was
repeated with the same conditions (400g (~1000 rpm) for 5 minutes) to remove the
excess dye. All the pellets were resuspended with 500 μl 1X Assay Buffer and 150 μl
from each of them was seeded into black 96-well plate as triplicate. While the aggregate
red form of the dye has absorption/emission maxima of 585/590 nm, the monomeric
green form of the dye released to the cytoplasm because of the loss of MMP has
absorption/emission maxima of 510/527 nm. The plate was read in these wavelengths
by fluorescence Elisa reader (Thermo Varioskan Spectrum, Finland). Finally, green/red
(510/585) values were calculated to determine the changes in MMP.
3.2.7. Total Protein Extraction from MM U-266 Cells
So as to extract the total proteins belonging to MM U-266 cells and 17 nM
(which is the IC-50 value of Bortezomib on U-266 cells) Bortezomib applied U-266
cells, whole cell suspension was taken from tissue culture flask into a sterile falcon tube
and then centrifuged at 1000 rpm for 10 minutes at room temperature, separately. After
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