Fixation of HeLa-M cells expressing Halo and SNAP fusion proteins conjugated to ligands

Mitophagy is a tightly regulated mechanism in which components are sequentially recruited to a damaged mitochondrion in order to clear it by lysosomal degradation. Defects in mitophagy are thought to contribute to multiple human diseases, including neurodegenerative diseases; thus, further study of the pathway is imperative. Many of the molecular steps that comprise mitophagy were first investigated in the HeLa culture system before their translation to specific cell types. Here we developed a protocol for conjugating multiple mitophagy components exogenously expressed in HeLa cells to commercially available fluorescent ligands with the Halo (Promega) and SNAP (New England Biolabs) systems. We precisely control the time course of our investigation by fixing cells with paraformaldehyde after timed incubation with a mitochondrial poison that induces global mitophagy. After fixation, cells can be imaged for a variety of assays and quantifications.