Supplementary Material for: Comparative Assessment of Mitochondria Isolation Buffers for Optimizing Tissue-Specific Yields in Buffalo

Introduction: Mitochondrial studies are crucial for assessing livestock health and performance. While extensive research has been done on cattle and pigs, the influence of mitochondria in Indian buffalo remains unexplored. Therefore, in order to understand functions of mitochondria, their energy-related processes, or any additional mitochondrial traits in buffaloes’; it is imperative to isolate high-yield mitochondria with purity and functionality. Mitochondria are extracted by few conventional buffers. These buffers were previously characterized for their effectiveness in isolating mitochondria from rodent and human tissues. Therefore, the present study is to assess the performance of mitochondria isolation buffers specifically in buffalo tissues.
Methods: The study involved isolation of mitochondria from four different tissues i.e. liver, brain, heart and muscles of slaughtered buffalo (n= 3), using: (i) Tris-Mannitol buffer (ii) Tris-Sucrose buffer and (iii) MOPS-Sucrose buffer. Buffer efficiency in preserving high fidelity during mitochondria isolation was assessed by comparison with Cayman's MitoCheck® Mitochondrial Isolation Kit (control). Further mitochondrial purity and functionality was assessed through comparative estimation of protein concentration and marker enzyme assays respectively.
Results: Our results revealed insights into the suitability of specific buffer for functional mitochondria isolation from specific type of buffalo tissue. Notably for obtaining high quality functional mitochondria from buffalo, MOPS-Sucrose buffer appeared optimal for soft tissues (liver and brain), while Tris-Mannitol buffer was efficient for hard tissues (muscles and heart).
Conclusions: Thus, Our research highlights the influence of buffer composition and tissue-specific variations in buffer effectiveness on mitochondrial activity in different tissues, leading to improved mitochondrial isolation in buffalo.