Abstract
Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated.
Highlights
IntroductionA programmed cell death, has a vital role in various cellular processes
Apoptosis, a programmed cell death, has a vital role in various cellular processes
The presence of inner-membrane phosphatidylserine (PS) on the outer membrane of a cell is one of the earliest events in apoptosis. This process can be monitored by Annexin V, which binds to negatively charged PS, in conjunction with 7AAD that has strong affinity for DNA and is efficiently excluded by intact cells
Summary
A programmed cell death, has a vital role in various cellular processes. Abnormal rate of apoptosis is closely associated with the pathophysiology of many diseases including cancer, autoimmune disorders and neurodegenerative disorders [1]. Apoptotic cells exhibits morphological and biochemical changes; at most lipids, proteins and DNA molecules are significantly perturbed. A change in the plasma membrane is one of the earliest responses to apoptosis, caused by translocation of phosphatidylserine from the inner to the outer leaflet of lipid bilayer. Existence of changes in proteins (e.g., caspase activation) and DNA fragmentation are other hallmarks of apoptosis [1,2]. Detection of apoptosis involves a variety of biochemical assays (caspase assays, mitochondrial dyes, DNA laddering technique) and flow cytometric analysis [1].
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