Adult Mouse Cardiac Myocyte Isolation - Solutions
Solutions (filter solutions #1, 2 & 3 using a 0.22mm mesh)
1. Ca2+ Tyrode: pH = 7.40 @ 37 C; 7.45 @ 25 C
| mM | M.W. | g/L | x10 | |
|---|---|---|---|---|
| NaCl | 137 | 58.44 | 8.006 | 80.06g |
| KCl | 5.4 | 74.55 | 0.403 | 4.03g |
| CaCl2 | 1.2 | 111.0 | 0.133 | 2.22g |
| MgCl2 | 0.5 | 203.3 | 0.102 | 1.02g |
| HEPES | 10 | 238.3 | 2.383 | 23.83g |
| Glucose | 10 | 180.2 | 1.802 | 18.02g |
2. Ca2+ free Tyrode: pH = 7.20 @ 37 C; 7.25 @ 25 C
| mM | M.W. | g/L | x10 | |
|---|---|---|---|---|
| NaCl | 135 | 58.44 | 7.889 | 78.89g |
| KCl | 4.0 | 74.55 | 0.298 | 2.98g |
| MgCl2 | 1.0 | 203.3 | 0.203 | 2.03g |
| HEPES | 10 | 238.3 | 2.383 | 23.83g |
| NaH2PO4 | 0.33 | 137.99 | 0.046 | 0.46g |
| Glucose | 10 | 180.2 | 1.802 | 18.02g |
| BDM* | 10 | 101.1 | 1.011 | 10.11g |
3. Enzyme Solution:
Mix up 75 ml of enzyme solution using Ca2+ free Tyrode with 25 mg of collagenase B, 20 mg of collagenase D and 5 mg of protease XIV.
4. Calcium Buffers:
0.06 mM 0.2 mM 0.6 mM 1.2 mM
Ca2+ free Tyrode 9.5 ml 8.3 ml 5.0 ml 0
w/ albumin
Ca2+ Tyrode 0.5 ml 1.7 ml 5.0 ml 10 ml
For these solutions mixup 75 ml of Ca2+ free Tyrode with 1mg/ml of albumin (Sigma; bovine serum albumin, fraction V). Use the remaining solution for rinsing and mincing the heart after enzyme digestion.
*BDM = 2,3-butanedione monoxime (Sigma)
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