Serum starvation: <i>caveat emptor</i>

TLDR

Serum starvation is widely used in molecular biology, yet protocols lack standard terminology, detailed procedures, and a common conceptual basis, leading to inconsistent results and questionable assumptions about reduced basal activity. The study aimed to investigate how serum starvation affects signaling pathways in primary human myotubes, rat L6 myotubes, and HEK293 cells under varied conditions. We examined time‑dependent changes in ERK1/2, AMPK, and mTOR signaling across different cell types and serum‑starvation protocols. Serum starvation provoked rapid, cell‑type‑specific changes in ERK1/2, AMPK, and mTOR signaling, with no uniform reduction in basal activity, underscoring its potential to confound experimental outcomes.

Abstract

Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.