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