If necessary, adjust the pH down to 7.4 with HCl. The final solution should still be clear. Now add the calcium and the rest of the substrates. The final perfusate should still be clear and not at all cloudy. It is now ready for filtration.
Filtration is vital before using the perfusate.
This is most easily accomplished by using a peristaltic pump and a two-part 47mm filter housing such as those from Millipore. In this manner, the perfusate can be filtered down to 1.2 microns in just one pass. We use the perfusate within 48 hours because of the instability of the acetoacetate. Copyright 2001 University of Texas Southwestern Medical Center at Dallas, All Rights Reserved.