Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry

Robin Rajan, Magdalena Karbowniczek, Haley R. Pugsley, Manoj K. Sabnani, Aristotelis Astreinidis, Ninh M. La-Beck

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Autophagy dysregulation has been implicated in numerous diseases and many therapeutic agents are known to modulate this pathway. Therefore, the ability to accurately monitor autophagy is critical to understanding its role in the pathogenesis and treatment of many diseases. Recently an imaging flow cytometry method measuring colocalization of microtubule associated protein 1B light chain 3 (LC3) and lysosomal signals via Bright Detail Similarity (BDS) was proposed which enabled evaluation of autophagic processing. However, since BDS only evaluates colocalization of LC3 and lysosomal signals, the number of autophagy organelles was not taken into account. We found that in cells classified as having Low BDS, there was a large degree of variability in accumulation of autophagosomes. Therefore, we developed a new approach wherein BDS was combined with number of LC3+ puncta, which enabled us to distinguish between cells having very few autophagy organelles versus cells with accumulation of autophagosomes or autolysosomes. Using this method, we were able to distinguish and quantify autophagosomes and autolysosomes in breast cancer cells cultured under basal conditions, with inhibition of autophagy using chloroquine, and with induction of autophagy using amino acid starvation. This technique yields additional insight into autophagy processing making it a useful supplement to current techniques.

Original languageEnglish (US)
Pages (from-to)451-458
Number of pages8
JournalCytometry Part A
Volume87
Issue number5
DOIs
StatePublished - May 1 2015

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Autophagy
Flow Cytometry
Light
Organelles
Chloroquine
Starvation
Autophagosomes
Cultured Cells
Breast Neoplasms
Amino Acids

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

Cite this

Rajan, R., Karbowniczek, M., Pugsley, H. R., Sabnani, M. K., Astreinidis, A., & La-Beck, N. M. (2015). Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry. Cytometry Part A, 87(5), 451-458. https://doi.org/10.1002/cyto.a.22652

Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry. / Rajan, Robin; Karbowniczek, Magdalena; Pugsley, Haley R.; Sabnani, Manoj K.; Astreinidis, Aristotelis; La-Beck, Ninh M.

In: Cytometry Part A, Vol. 87, No. 5, 01.05.2015, p. 451-458.

Research output: Contribution to journalArticle

Rajan, R, Karbowniczek, M, Pugsley, HR, Sabnani, MK, Astreinidis, A & La-Beck, NM 2015, 'Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry', Cytometry Part A, vol. 87, no. 5, pp. 451-458. https://doi.org/10.1002/cyto.a.22652
Rajan, Robin ; Karbowniczek, Magdalena ; Pugsley, Haley R. ; Sabnani, Manoj K. ; Astreinidis, Aristotelis ; La-Beck, Ninh M. / Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry. In: Cytometry Part A. 2015 ; Vol. 87, No. 5. pp. 451-458.
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