We perform primary cardiomyocyte isolation from 2-3 day old rat neonates. We recurrently obtained a cell yield of ~20 Mil cells from a litter of 10 pups. But, since the last 6 months, the cell yield has been almost reduced to half even though the same protocol is being followed. We have tried different collagenase enzyme, fresh trypsin and media but to no avail.
Could someone please suggest what other factors we could optimize and improve?
Thanks Jean,
We follow slightly different protocol - we isolate the hearts and place it in cold HBSS (without Ca & Mg) after dissecting into small pieces, the tissue pieces is incubated in 4C trypsin overnight. The trypsin activity is stopped with DMEM media (with phenol red, 10% FBS and 5% Penstrep). This is then followed by 2 sequential stirring (heavy) in warm 37C collagenase.
The rest is as you said, collection of the supernatant form the last stirring and pre-plating.
This protocol gave as really good yield until end of summer, but since then we haven't been able to pin-point any cause that drastically reduces the yield to less than half, when compared to our original result!
thank you Professor Gomez,,, would it make any difference if we have to use mice instead of rat?
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