%0 Journal Article
%T A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization.
%A Gautam N
%A Sankaran S
%A Yason JA
%A Tan KSW
%A Gascoigne NRJ
%J Methods
%V 134
%N 0
%D 02 2018 1
%M 29198814
%F 4.647
%R 10.1016/j.ymeth.2017.11.015
%X 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 naïve, 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.