Primary cardiac fibroblast and cardiomyocyte isolation

1. Ventricles were removed under sterile conditions.
2. Ventricles were placed in cold sterile primary cell culture medium, minced into approximately 2 mm cubes and treated with solution of primary cell isolation.
3. Tissue fragments retained on 10⁵ μm nylon mesh were placed in primary cell culture medium containing 1% bovine serum albumin (BSA) and 100 μM CaCl2, teased apart with sterile forceps and dispersed by trituration using a wide-bore pipette.
4. Tissue fragments were removed and the cell suspension was adjusted to 500 μM CaCl_² and centrifuged over a 5 ml cushion of primary cell culture medium containing 6% BSA.
5. The resulting cardiomyocyte-enriched pellet was resuspended in primary cell culture medium containing primary cell culture supplement and 10% fetal bovine serum and seeded on 100 mm collagen-coated tissue culture plates for 3 h at 37 °C in a CO_² incubator to remove non-myocyte cells.
6. Non-adhered cardiomyocytes were collected and stored at −80 °C.
7. For cardiac fibroblast isolations, ventricles were removed as noted above, minced and incubated in Hank's buffer containing trypsin (0.1 mg/ml) and collagenase (50 units/ml) for 10 consecutive 10 min treatment periods at 37 °C.
8. Cells from each digestion period were pooled, resuspended in primary cell culture medium containing primary cell culture supplement and 10% FBS and seeded in standard culture dishes for 8 h.
9. Non-adherent debris was discarded and the attached fibroblasts were maintained in primary cell culture medium incuding 10% FBS, scraped into ice-cold PBS, pelleted and stored at −80 °C for EMSA and Western blot analysis.

Reference
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