Hi there, I'm having trouble with my Hl-1 cardiomyocytes that do not spontaneously beat. I do strictly follow Claycomb's instructions about culturing (e.g. maintenance, splitting, digestion with trypsin, passaging and so on). Has anyone else ever experienced something like this?
Dear Andrea,
I have never worked with HL1 cells but i think that these cells become contractile only when they are at confluence and that after a number of passages they loose this ability.
In addition, the original HL-1 line is not very contractile. You can try to sub-clone the original line and search for more contractile sub-clones. Try to dilute them and some colonies will be contracting, then take them out using cloning cylinder.
Hope it helps. Best wishes,
Marc
Dear Marc, thanks for your reply.
I did try to search for any sign of contraction when the cells were 100% confluent, as reported by many papers. No luck and thus no chance of subcloning them.
Also, this problem does not seem dependent by the passage number of the cells. My first thought was that the batch I'm working with lost somehow the cardiac phenotype. However, I can still measure nice spontaneous Ca2+ transients. Fluo-4 loaded HL-1 cells respond as expected to isoproteranol, high K+, carbachol and so on. In addition, ipertrophic response to known stimuli is normal.
Am I missing something? Contractile proteins?
Thanks again Marc, have a good day!
Andrea
Dear Andrea,
Although they may have lost their contractile properties, they may still have normal electrical and calcium activities.
As I said try to dilute them and some colonies will be contracting then take them out using cloning cylinder. But if you have tried and it has not worked out, I done know what may be happening. I don't see anything that you are possibly missing.
Sorry for not being very helpful. Best of lucks!!!
Marc
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