Hello Hemi.

I think that your collection procedure of cardiac cells should be properly excecuted. Tyrysin-EDTA could only collect non-cardiomyocytes or non-cardioblasts deep within the heart tissues.

Follow appropriate articles and references within the article and study more. Once you have proper cell recovery, immunocytochemical staining is not a problem.

Here's one you may start with.

- Isolation and culture of neonatal mouse cardiomyocytes (2013)

- Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy (2014)

Regards.

Hi Sang,

Thanks for your reply.

I'm sorry for the confusion, but i actually did perform the isolation properly (I used Pierce Primary Cardiomyocyte Isolation Kit), and cultured the cells for 3 days.

The cells were fine, and they were contracting nicely, before I harvested them from the dish with trypsin. my concern about trypsin is due to the fact that Cn-43 is partly a membranal protein, thus may be affected by it, but i did see in several papers that this was done.

Can you tell me which antibody did you use for rat Cn-43?

Many thanks

Hemi

Hello Hemi.

Sorry for my misunderstanding. Even the isolated cardiac cells culture in vitro could still synthesize and secret collagen as an ECM. Thus, cells are most likely to be lysed or damaged only by trypsin to try to disperse into single cell suspension. Incubate the cell dish containing Hank's BSS with the same collagenase as you used in isolation cells from the heart in CO2 incubator for 20 min after washing the medium with PBS. Then use 1/5 dilution of trypsin-EDTA with only whirling without using pipetting. This will save the Cx43 expressed on your cells without cell damage.

Best wishes.