METHODOLOGICAL APPROACHES TO DETECTION AUTOPHAGY IN MUSCLE CELLS

Abstract

Introduction. Autophagy is a process, whereby components of cell undergo degradation influenced by lysosomal enzymes leading to autophagosome formation. Nowadays, autophagy is considered as one of the application point in range of pharmological approaches, particularly, in inherited orphan diseases and also in aging, and heart failure. O bject of research and properties of examined cell culture have the essential meaning for methodological evaluation of autophagy. In this work, we present selection of optimal conditions for evaluation of autophagy progress in C2C12 cell line.

Methods. To select the optimal approach to evaluate autophagy progress, we tested flow cytometry, immunocytochemistry, and immunoblot (Western blot). T o choose the optimal conditions of cell membrane permeabilization performance various detergents, temperature conditions, time of cell fixation, and components of lysis buffer were compared.

Results. We report, that application of 0,025% digitonin is decisive condition to extract separate protein LC3 fractions (soluble and insoluble). Samples fixation and reduced time of washing are necessary for flow cytometry and allow to maintain optimal number of cells for analysis based on detergent application. We consider, that applying of cell transfection with constructs, carrying LC3, can lead to increase the required time of serum deprivation.

Conclusions. O ptimized protocols for evaluation of autophagy in C2C12 cells over detection of separate fractions of LC3 can be effectively used in investigation of fundamental molecular and cellular mechanisms of myopaties and cardiomyopaties development.