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FAQ:
1. Can I use Langendorf perfusion system
with Adumyts kit?
Yes! You can use Langendorf perfusion system with Adumyts kit
together and set the temperature at 37° C as well. It is not
necessary to bubble the Adumyts kit solutions with oxygen gas. It is
very important to use stabilization buffer included in the kit.
2. Is the SureCoat solution reusable?
The SureCoat solution can be used only once because of possible
degradation of some adhesive proteins.
3. What may go wrong if the adult rat or
mouse heart is not digested well or if I can not see clear
striations on cells under the microscope.
It is very important to perform the following sequential perfusions:
After perfusion with 50 ml of B1 solution, add the 50 ml of B2
solution (that does not contain stabilization buffer). Collect 1st
time B2 solution and add 100 ul of stabilization buffer for the 2nd
time perfusion. Collect 2nd time B2 solution and add 100 ul of
Stabilization buffer for the 3rd perfusion, Then collect 3rd time B2
solution and 100 ul of stabilization buffer for the 4th The normal,
healthy, viable rat or mouse cardiomyocytes are rod-shaped cells
with clear striations. Before performing the perfusion, You must
first identify the aorta. The aorta has little artery branches on
it. When you excise the heart from rat or mouse, leave the aorta at
least 0.5 cm above aortic valve so that you can easily cannulate it
onto the perfusion system. Additionally, you must make sure that
cannulating tubing is placed above the openings of coronary arteries
to avoid the blockade of coronary perfusion.
4. How can I improve the isolation process
when I get low yield of both adult and neonatal
cardiomyocytes?
You may consider quick removal of digestion buffer from
disassociated cells. Transfer each digestion to sterile tube, spin
down, and resuspend cell pellet in storage solution. Make sure that
the heart tissue is completely digested. You may only see the
connective tissues left at the final step of digestion.
5. I seem to get good yield, but the cell
viability is low from isolation of both adult and neonatal rat or
mouse cardiomyocytes.
Check the digestion time to see if it is longer than the protocol.
Avoid over digestion. Remove as much supernatant as possible.. The
longer cells are exposed to the digestion buffer, the less viable
cells are obtained.
6. After 24 hours of culture at 37 C, a lot
of cells are floating on the plates. Why don't they attach to the
plates?
Coat the plates for at least one hour in the 37° C oven or
incubator. We recommend to use our SureCoat solution and culture
medium which are optimized for cardiomyocyte cultures. Also, make
sure to avoid over digestion and harvest as many healthy
cardiomyocytes as you can.
7. What percentage of cardiac fibroblasts
are present in cardiomyocyte cultures?
To get 100 % pure cardiomyocytes is really hard. However, our
isolation systems guarantee our customers to get > 97 % pure
cardiomyocytes. To check the cell purity, the most effective and
convenient method is to perform immunocytochemical staining of
sarcomeric alpha actin, which is a unique marker of cardiomyocytes
and does not exist in cardiac fibroblasts. The staining protocol is:
Here.
Still have questions? No problems, our technical support
is here:
techsupport@cellutron.com
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