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Issue 12, 2014

Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Author affiliations

Abstract

Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal evolution of multicellular organisms. Its misregulation has been associated with many diseases, making its early and reliable detection a key point for modern cellular biology. In this paper, we propose the use of infrared microspectroscopy (IRMS) as a label-free methodology for the detection of apoptotic-related biochemical processes induced on U937 leukemic monocytes by serum starvation and CCCP-exposure. The spectroscopic results are in agreement with parallel Flow Cytometry (FC) experiments, where plasma membrane integrity and mitochondrial activity were assessed. Spectroscopic outcomes complement FC data and allow drawing a more complete picture of the apoptotic pathways. In particular, we established that the two apoptosis-inducing treatments, cell starvation and CCCP exposure, affect the cell cycle in a different way. With the former, cell death is preceded by a cell cycle arrest, whereas the latter causes an increased cell cycle progression. Spectral data demonstrate that for both conditions apoptosis proceeds through the accumulation of lipid droplets within cells. Moreover, we were able to establish a spectral marker for DNA condensation/fragmentation: the enhancement of the PhI band component centred at ∼1206 cm−1, which is more sensitive than the relative intensity of the PhII band to which phospholipids and carbohydrates also contribute significantly. In conclusion, we demonstrate that the intrinsic multi-parametric nature of IRMS and its application on cells under physiological conditions can be well exploited for the investigation of apoptotic pathways.

Graphical abstract: Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Article information


Submitted
13 Feb 2014
Accepted
27 Mar 2014
First published
28 Mar 2014

This article is Open Access

Analyst, 2014,139, 3097-3106
Article type
Paper
Author version available

Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

G. Birarda, D. E. Bedolla, E. Mitri, S. Pacor, G. Grenci and L. Vaccari, Analyst, 2014, 139, 3097 DOI: 10.1039/C4AN00317A

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