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 > 95 % 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
 

Home