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Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells
Published on: January 9, 2019
High-Resolution Multicolor Imaging of Mitochondria in Lymphocytes
Munir Akkaya1, Pietro Miozzo2, Margery G Smelkinson3
1Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. munir.akkaya@nih.gov.
Insights
Researchers developed a new microscopy method to visualize mitochondrial changes in immune cells during activation. This technique aids the study of immunometabolism by improving imaging of lymphocyte mitochondrial morphology.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Cellular metabolic states change during immune activation, a key area in immunometabolism.
- Visualizing mitochondrial organization is crucial for studying these metabolic shifts.
- Existing imaging methods are not ideal for many immune cells, particularly lymphocytes.
Purpose of the Study:
- To develop a novel multicolor, high-resolution microscopy strategy for imaging mitochondrial morphology in lymphocytes.
- To adapt current imaging techniques for immune cells, overcoming limitations of previous methods.
Main Methods:
- Devised a multicolor high-resolution microscopy strategy.
- Utilized TOM-20 staining for the mitochondrial surface and Mitotracker dyes for the mitochondrial matrix.
- Included methods for efficient exclusion of nonviable cells.
Main Results:
- Successfully imaged mitochondrial morphology in both resting and activated lymphocytes.
- The method allows simultaneous staining of mitochondrial surface and matrix.
- Demonstrated the exclusion of nonviable cells from imaging.
Conclusions:
- The novel imaging strategy is effective for studying mitochondrial morphology in lymphocytes.
- This technique provides a valuable tool for research in immunometabolism and lymphocyte activation.
- Complements existing research on lymphocyte metabolism and mitochondrial function.
Abstract:
The identification of cellular changes that accompany immune activation has been a long-standing interest for immunologists. Among these, alterations in the metabolic states of these cells have gained particular attention in the last decade due to the emergence of the field of immunometabolism. A thorough investigation of these metabolic changes can only be achieved with an in-depth visualization of mitochondrial organization; however, current strategies for mitochondrial imaging have been optimized in model cells with a high cytoplasm-to-nucleus ratio and thus are not readily adaptable for many immune cells. Here, we devised a multicolor high-resolution microscopy strategy to image mitochondrial morphology in lymphocytes at both their resting and activated states. Our method allowed us to stain both the mitochondrial surface (by targeting TOM-20) and the mitochondrial matrix (through the use of Mitotracker dyes) while efficiently excluding nonviable cells. Our novel imaging strategy offers a powerful tool to study changes in mitochondrial morphology and complements any research focusing on lymphocyte metabolism.
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