We are currently conducting force measurements on electrically (approx. 25 Hz/10 ms, 4-5 V for 1 sec) stimulated mouse hind limb muscle (EDL, soleus) suspended in carbogen-bubbled, bicarbonate-buffered physiological solution (in mM):
137 NaCl, 5 KCl, 24 NaHCO3, 1 NaH2PO4, 11 dextrose, 1 MgSO4, 2 CaCl2 at 37C.
Part of the experiments involves testing of precious, scarce compounds, which would be hard to bubble, so we have thought of simply exchanging the bath solution with stagnant [bubbled control solution + drug]. This, however, worries me because any experiment extending beyond a couple minutes will, we believe, see an increase in pH as the bath solution is no longer CO2-equilibrated. We are, therefore, considering replacing the bicarbonate (with the exception of maybe 1 mM) with 10 mM HEPES. Given the reduction of dissolved salts, osmolality will have to be adjusted as well, for which we could use sucrose, I suppose (approx. 4.45 g/l).
Questions:
(1) Any there any concerns regarding the HEPES substitution (or removal of almost all bicarbonate) that could significantly compromise the physiology of the muscle?
(2) If we have to stick with the bicarbonate buffering approach, how long before the start of the experiment should we begin bubbling? How long after the bubbling is terminated does the solution remain acceptably constant in terms of pH?
Thank you.
I would stick with the traditional bicarbonate buffer system. Whether stopping the bubbling for a couple of minutes makes any difference really depends on the volume of bathing solution compared with the volume of tissue. What volume of fluid do you use? You have two problems, one is the buffering but the other one is oxygenation and while replacing changing the buffer may solve the need for CO2, the other problem is keeping the solution fully oxygenated, which really needs bubbling with O2.
Why not just try it out.. stop the bubbling for 2 minutes and see how your preparation behaves, does it produce the same force and recover as well as when you bubble it?
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