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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3.1. Materials<br />
3.1.1. Medias<br />
CHAPTER 3<br />
MATERIALS AND METHODS<br />
Medias were listed <strong>in</strong> Appendix A.<br />
3.1.2. Chemicals, Reagents and Solutions<br />
Reagents and solutions were presented <strong>in</strong> Appendix B.<br />
3.2. Methods<br />
3.2.1. Cell L<strong>in</strong>es and Culture Conditions<br />
U-266 human MM cells were k<strong>in</strong>dly obta<strong>in</strong>ed from the German Collection of<br />
Microorganisms and Cell Cultures (Germany). The cells were grown and ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong><br />
RPMI-1640 growth medium conta<strong>in</strong><strong>in</strong>g 10% fetal bov<strong>in</strong>e serum (FBS) and 1%<br />
penicill<strong>in</strong>-streptomyc<strong>in</strong> (Invitrogen) at 37°C <strong>in</strong> 5% CO2. Medium was refreshed every 3<br />
days. In order to passage the cells, whole cell suspension was taken from tissue culture<br />
flask (75cm 2 ) <strong>in</strong>to a sterile falcon tube (50ml) and then centrifuged at 1000 rpm (~800g)<br />
for 10 m<strong>in</strong>utes (m<strong>in</strong>) at room temperature. After centrifugation, the supernatant was<br />
removed and the pellet was washed with 3-4 mL Phosphate Buffered Sal<strong>in</strong>e (PBS) and<br />
centrifugation process was repeated. In the follow<strong>in</strong>g step, supernatant was removed<br />
from the tube and the pellet was resuspended with 15 mL of RPMI-1640 (10% FBS and<br />
1% Penicill<strong>in</strong>-Streptomyc<strong>in</strong>). Then, it was transferred <strong>in</strong>to a sterile 75cm 2 filtered tissue<br />
culture flask and <strong>in</strong>cubated <strong>in</strong> CO2 <strong>in</strong>cubator.<br />
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