I work with neonatal rat ventricular cardiomyocytes, and I want to do calcium imaging of the same. I have been trying to attach cardiomyocytes to glass coverslips and I have tried a few substrates like laminin, fibronectin, collagen and gelatin. But the cells in the glass coverslips detach or die on the second day of the culture whereas those in dishes remain healthy. Has anyone tried attaching the cardiomyocytes to glass coverslips? Could you suggest to me a substrate?
In case of Fibronectin, a counter-intuitive reality is that you must wash the coverslips well in PBS after incubating with FN. No experience with these cells, but FN is always my first choice
Fibronectin according to this paper seems working!
http:/ /ajpheart.physiology.org/content/285/1/H325.full
However, I deal with renal epithelial cells and poly d lysine works well for mine.
If in the second day your neonatal cardiomyocytes are dead I think it's because of culture medium. Laminin for attaching and DMEM/F12 + 20%FBS + L-glutamine and antibiotics as medium. Also I suggest chamber slides better than coverslides although it's working as well.
We routinely use coverslips for immunofluoreescnece experiments in neonatal rat cardiomyocytes. We use coverslips size of wells of 12 well plate. We use Collagen solution (Type 1 from calf skin, Sigma, C8919) for coating the coverslips for 5-6h, followed by three washes with water and drying O/N in culture hood. Number of cells we put in each well is 200,000 / well and at the end we get quite a good number of adherent cells even after 72h of virus infection.
Thanks to everyone for the suggestions. Nuria Gago-Lopez- I dont think the problem is with the culture medium. Because the cells from the same prep that are in culture dishes remain healthy.
Ashraf Rangrez- I used collagen this time, I coated the coverslips only for an hour. Although the yield was less, the cells looked better. Next time I shall try 6h coating and drying it overnight.
In my experience 1% gelatin coating on glass chamber slide is enough to obtain a good adhesion of mice primary cardiomyocyte, who remain alive for at least a week. However I noticed some morphological differences between cell seeded on plastic and on glass both of them with gelatin coating.
I have also trouble with this problem, I used collagen coating for at least 4 hours, and wash three times with DPBS, drying it at hood for 1 hour. But there is no cell adherent to the glass, meanwhile, the cells in the dish still healthy. I can't find a solution. Could you help me?
Min Li,
If you use neonatal cardiomyocytes collagen would be fine and if you use adult cardiomyocytes, laminin would be best option. Becasue of the hydrophobicity of the glass water/buffer would not form a thin layer. So repeated pippeting until it form a layer on glass would work.
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