The Importance Of Culture Medium And FDB In The Isolation Of Single Fdb Fibers Using A Collagenase Digestion

Abstract

Most studies on muscle fatigue use whole muscle. The disadvantages of such preparations are the development of an anoxic core and the impossibility of studying differences between fiber types. While some studies have used mechanically dissected single muscle fibers, the preparation is time consuming and difficult to use for electrophysiological measurements. Another method is to isolate single fibers in large numbers following a collagenase treatment to allow for dispersal of fibers by trituration. Although variations on the method to obtain such single fiber preparations exist, there is no quantitative data indicating quality of preparation in terms of contractility. The objective of this study was to determine the best protocol for fiber isolation from mouse FDB fibers. Fibers isolated following collagenase digestion in MEM culture medium with 10% FBS had better morphology and contractility (i.e., number of contracting fibers and threshold values) than those in which DMEM was used as a culture medium. Also, in the absence of FBS in culture medium, all fibers supercontracted during trituration regardless of the culture medium used. The addition of 0.2% FBS in the physiological solution bathing fibers during experiments also improved morphology, contractility and stability.