I have recently established an HL-1 cardiomyocyte cell line from a frozen aliquot. I don't see the cells contract in the culture. I have already passaged them 4-5 times. I want to study calcium channels in these cells. Should I be concerned as they are not contracting?
I'm not an expert of 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.
The original HL-1 lines is not very contractile, Aurelien is right. But they probably still have action potentials and calcium transients. We sub cloned the original line and obtained clones that are more contractile although, you need to freeze them regularly because they loose gradually this property but they still have normal electrical and calcium activities. Have a look at this paper we recently got out: http://www.ncbi.nlm.nih.gov/pubmed/24587307,
Characterisation of connexin expression and electrophysiological properties in stable clones of the HL-1 myocyte cell line.Dias P, Desplantez T, El-Harasis MA, Chowdhury RA, Ullrich ND, Cabestrero de Diego A, Peters NS, Severs NJ, MacLeod KT, Dupont E.
PLoS One. 2014 Feb 28;9(2):e90266. doi: 10.1371/journal.pone.0090266. eCollection 2014.
Try to dilute them and some colonies will be contracting then take them out using cloning cylinder.
Good luck
Emman
Thanks Emmanuel. I just wanted a clarification from the paper. What was the composition of the lysis buffer you used to lyse the cells for protein prep? Did you just use 20% SDS solution? I am planning to do a western in the near future from HL-1 cells and looking for the best and hassle free method out there.
It contain also 100 mM tris pH 6.8 and 10 mM EDTA, use 10ul/cm2 of confluent monolayers this should give you about 5ug/ml prot. I use the bio-rad DC prot assay after diluting 20x (1%SDS final conc) and do your standard curve (IgG or BSA) in the same solution. You will need to sonicate to break the DNA otherwise it's very viscous. Even a small volume (50ul) with a microtip. Remember to add 2 ME to 2.5% before electrophoresis and make sure you load ALL the wells even without protein because 20% SDS make the lanes expands. These prot solutions are very stable.
Cheers
Emman
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