Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations

Int J Mol Sci. 2020 Oct 26;21(21):7944. doi: 10.3390/ijms21217944.


Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

Keywords: astrocytes; enzymatic digestion; microglia; microglia isolation; neurons; protocol; single-cell sequencing.

MeSH terms

  • Animals
  • Astrocytes / chemistry*
  • Brain Neoplasms / genetics
  • Brain Neoplasms / metabolism*
  • Cell Line, Tumor
  • Cell Separation / methods
  • Chromatography, Liquid
  • Enzymes / metabolism*
  • Flow Cytometry / methods*
  • Gene Expression Profiling / methods*
  • Glioma / genetics
  • Glioma / metabolism*
  • Humans
  • Male
  • Mice
  • Microglia / chemistry*
  • Neoplasm Transplantation
  • Proteomics / methods
  • Sequence Analysis, RNA
  • Single-Cell Analysis
  • Tandem Mass Spectrometry


  • Enzymes