Journal
METHODS
Volume 134, Issue -, Pages 11-19Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2017.11.015
Keywords
T cells; Mitochondria; MitoTracker Green FM; Imaging flow cytometry (IFC); Amnis ImageStream; Raw Max Pixel
Funding
- Singapore Ministry of Health's National Medical Research Council [CBRG15-may017]
- Singapore Ministry of Education's grant [2014T2-1-136]
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Mitochondria, the powerhouse of the cell, are known to remodel their membrane structures through the process of fusion or fission. Studies have indicated that T cells adopt different energy metabolic phenotypes, namely oxidative phosphorylation and glycolysis depending on whether they are naive, effector and memory T cells. It has recently been shown that changes in mitochondrial morphology dictate T cell fate via regulation of their metabolism. Our keen interest in T cell function and metabolism led us to explore and establish a method to study mitochondria in live T cells through a novel high content approach called Imaging Flow Cytometry (IFC). The focus of our current study was on developing a protocol to standardize the concentration of MitoTracker Green FM dye to observe mitochondria in live T cells using IFC. We began the study by using widefield microscopy to confirm the localisation of MitoTracker Green FM labelled mitochondria in live T cells. This was followed by testing various concentrations of the dye to achieve a similar labelling pattern using IFC while eliminating false positive or negative staining. The optimization of the method used to label the mitochondria by IFC for analysis included standardisation of a number of important parameters such as dye concentration, voltage, fluorescence intensity values for acquisition and processing. IFC could potentially be a powerful method to study T cells in a relatively high throughput manner. (C) 2017 Elsevier Inc. All rights reserved.
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