Title:Flow Cytometric Analysis of Protein Aggregates
VOLUME: 24 ISSUE: 10
Author(s):Sushanta Debnath, Bikram Nath and Abhijit Chakrabarti*
Affiliation:Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata
Keywords:Scattering intensity, autofluorescence, amyloid aggregates, nanoparticles, proteins, flow cytometer.
Abstract:Background: Misfolding of proteins often leads to aggregation. Accumulation of diverse
protein aggregates in various cells, tissue and organs is the hallmark of many diseases, such
as Alzheimer's disease and Parkinson's disease.
Objectives: The main objective of this study was to present a novel method of characterization of
protein aggregates, associated with differential toxicity with different size and composition in vitro
using flow cytometry.
Methods: A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm
was used for flow cytometry analysis. The voltage and the gain settings for individual channels
were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed
Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of
proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of
aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using
Flowing software version 2.5.1 and Origin 8.0.
Results: Autofluorescence and scattering intensities could distinguish between amyloid and nonamyloid
aggregates. Dot plots of both side scattering (SSC) and forward scattering (FSC) intensities
also showed characteristic fingerprint of both the types of aggregates when compared with those of
well known nanoparticles of oxides of Fe and Cu.
Conclusion: This work reports a novel, simple and robust flow cytometric method of characterization
of protein aggregates of different size and composition which would find wider application in
characterization of biomolecular aggregates, in general.
