practical Flashcards
coomassie stain?
Is a general protein stain that is mainly used to stain proteins in SDS-PAGE. The dye
interacts electrostatically but noncovalently with the amino and carboxyl groups of
proteins. Here we have used it to stain all the proteins in the cells.
the cancer cell lines
Cancer cell lines:
HeLa (cervical cancer)
H358 (lung cancer)
H358 are more aggressive; grow in clumps; will also grow well in soft agar assays
skeletal muscle cells
Skeletal muscle cells:
C2C12 GM
C2C12 DM
GM (growth medium): DMEM with 20% Fetal Calf Serum; leads to proliferation; DM
(differentiation medium): DMEM with 2% Horse Serum; leads to differentiation, i.e.
the single C2C12 myoblasts will fuse to myotubes and then start to assembly
myofibrils; there are only a few very short and stubby myotubes to be seen in these
samples (rest lifted off the glass coverslips); these are very dark blue and in the
slides that are marked with red exclamation marks
Question: What does staurosporine do? Which cell biological phenomenon do we
see here (was already covered in one of the lectures)?
Staurosporine is a broad band kinase inhibitor and induces apoptosis when cells are
exposed to it for longer time, because one of the many things it interferes with is cell
adhesion. The treated cells are starting to lift off the dishes and have developed
these very long processes, with occasionally visible death blebs
Describe differences in phalloidin, mitochondria and DAPI signals in C2C12
myotubes compared to myoblasts. How many nuclei per cell? Signal intensity for
mitochondria and phalloidin different between myotubes and myoblasts?
into myotubes (mature muscle cells), and how these changes can be visualized using fluorescent stains under a microscope. Here’s a breakdown:
🔹 Cell Differentiation
C2C12 myoblasts are mononucleated (one nucleus per cell).
When they differentiate, they fuse together to form multinucleated myotubes (mature muscle cells with multiple nuclei).
This fusion is visible because differentiated cells have multiple DAPI-stained nuclei per cell.
🔹 Stains and Signals
DAPI (blue signal):
Stains DNA (nuclei).
Shows how many nuclei are present per cell.
In differentiated cells, the nuclei may become more oval (ovoid) and resemble moons in shape.
Phalloidin (green signal):
Stains F-actin, a component of stress fibers in the cytoskeleton.
Stress fibers appear more intense and organized in differentiated myotubes.
MitoTracker (red signal) (or other mitochondrial stain):
Stains mitochondria, which appear worm-like.
Mitochondrial content increases in differentiated myotubes because these cells have higher energy demands compared to undifferentiated myoblasts.
