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

3.2.2. Thawing the Frozen Cells
Cells (1ml) were removed from frozen storage at -196°C (Liquid Nitrogen) and
quickly thawed in a water bath at 37°C so as to acquire the highest percentage of viable
cells. When the ice crystals melted, the content was immediately transferred into a
sterile filtered tissue culture flask (25cm 2 ) containing 5-6 ml of RPMI-1640 growth
medium and incubated overnight at 37°C in 5% CO2. After incubation, cells were
passaged as mentioned before.
3.2.3. Freeze the Cells
Cells taken from tissue culture flask were centrifuged at 1000 rpm for 10
minutes at room temperature. After centrifugation, the supernatant was carefully
removed and the pellet was resuspended by the addition of 4.5ml FBS and 0.5ml
dimethyl sulfoxide (DMSO). Then, gentle pipetting was applied and the cell suspension
was transferred to the cryogenic vials (1 ml) by labeling. At the following step, these
cryogenic vials were lifted to freezing compartment -80°C, for overnight by wrapping in
cotton wool in a polystyrene box to prevent the cells from shock. Finally, they were
transferred to liquid nitrogen dewar for long-term storage.
3.2.4. Cell Viability Assay
The assay primarily based on distinguishing dead cells from alive cells with the
addition of trypan blue dye. ~30 µl of trypan blue dye was mixed ~30 µl of cells (1:1
ratio, volume/volume) in order to measure the viability of cells. When cells were treated
with trypan blue dye, viable cells would be normally impermeable to it, whereas dead
cells would permeate by virtue of breakdown in membrane integrity. Thus, cells can be
observed as unstained cells that are alive or blue stained cells that are dead under a
microscope. By applying this assay, cells were counted using a hemocytometer in the
presence of trypan blue solution, under microscope. Then the percentage of viable cells
was calculated. Cell viability assay was conducted before each experiment.
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