So I am trying to isolate cardiomyocytes for calcium imaging purposes; however, I cannot get expected results with isoproterenol even at high concentration (1mM). It's definitely the cell condition that's the problem but I cannot pinpoint exactly which part to fix. Now I am concentrating on the storage solution (Kraft-Bruhe containing: 70 mM KOH, 0.5 mM EGTA, 10 mM HEPES, 50 mM KCl, 50 mM L-glutamic acid, 3 mM MgCl2, 20 mM taurin, 20 mM glucose, 20 mM KH2PO4). I also try to gradually increase calcium levels for calcium reintroduction at increments of 200 micromolar CaCl2. I also keep the cells in 4 dec C. There is no myocyte movement until addition of 200 to 400 uM calcium, but cell viability never reaches 70-80%. I am using wistar rats as subject, and collagenase (Yakult) alone for enzymatic digestion. Struggling MS degree candidate here. Any help will be greatly appreciated. Thank you!
Hi Julius, I think your isolated cells look quite well. But, as far as I understand your problem, after calcium reintroduction your get a problem with hypercontraction? I personally have made the experiance that cells are less sensitive to calcium when they have been satteld allready. And you might use BDM, which is used for contraction inhibition but it also reduces cellular calcium activity (that you can nicely observe in calcium imaging), thus it might prevent hypercontraction during your isolation procedure. Beside this, your glucose concentration appears a bit high, i would recommand to use 5 mM insted of 20 mM and 5 mM lactate.
Hello, Dr. Peetz! Thank you for the very helpful response! But what do you mean by settling the cells first before calcium reintroduction? If I understand it correctly, what I do is I keep the cells in KB at 4C for about 10 minutes, then I add 200 uM calcium. I add increments of 200 uM calcium cumulatively, at 10 minute intervals. Also, you might be right about the excessive glucose concentration I've been using, apparently I've been using twice as much compared to other references; however, this is a standard formulation my laboratory uses so I had to comply. But I will try it to see how it changes my cell condition. Again, thank you very much!
I see. However, in my case, I keep the cells in a cell suspension of KB as I use the cells within the day of isolation. I shall look into that as well. Thank you for the clarification!
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