Experiment Overview
| Repository ID: | FR-FCM-ZYKR | Experiment name: | A reproducible, non-biased method using MitoTracker® fluorescent dyes to assess mitochondrial mass in T cells by flow cytometry | MIFlowCyt score: | 83.12% |
| Primary researcher: | Genevieve Clutton | PI/manager: | Genevieve Clutton | Uploaded by: | Genevieve Clutton |
| Experiment dates: | 2017-02-01 - 2018-05-31 | Dataset uploaded: | May 2018 | Last updated: | Jun 2018 |
| Keywords: | [T-cells] [mitochondria] [Immunologic techniques] | Manuscripts: | [30576071] |
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| Organizations: |
University of North Carolina at Chapel Hill, Microbiology and Immunology, Chapel Hill, North Carolina (United States of America)
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| Purpose: | To optimize and validate a non-biased method to assess mitochondrial mass and polarization in CD4+ and CD8+ T cells using MitoTracker® dyes. Our method involves gating MitoTracker® high populations by fluorescence percentile. When analyzing data we first removed outliers, identified the fluorescence intensity of the brightest and dimmest events and then gated cells within the top 90% (above the 10th percentile) of this fluorescence range. Peripheral blood mononuclear cells (PBMC) were stained in triplicate with two aliquots of MitoTracker® dye from the same lot. We compared reproducibility between aliquots using our pecentile gating method and using median fluorescence intensity (MFI). | ||||
| Conclusion: | We demonstrate that gating MitoTracker® ‘high’ subsets by fluorescence percentile reduces variability between technical replicates compared with assessing MitoTracker® fluorescence by MFI. This is a non-biased method that will allow consistent results to be obtained when data are analyzed by different operators. | ||||
| Comments: | None | ||||
| Funding: | This study was supported by NIH NIAID grant U01 AI131310. Research reported in this publication was supported by the Center for AIDS Research award number 5P30AI050410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. | ||||
| Quality control: | Measurements were performed in triplicate (three technical replicates per dye aliquot). LSRFortessa performance was calibrated daily using CS&T Research Beads (BD Biosciences). Data were compensated using single-stained compensation controls. Fluorescence minus one controls were used to check for spectral overlap between fluorescence channels. | ||||
Experiment variables
| Doses | |
|---|---|
| · Aliquot 1 | 170718 01099 50 nM MTG + 15 nM MTDR vial 1_1_001.fcs · 170718 01099 50 nM MTG + 15 nM MTDR vial 1_2_002.fcs · 170718 01099 50 nM MTG + 15 nM MTDR vial 1_3_003.fcs |
| · aliquot 2 | 170718 01099 50 nM MTG + 15 nM MTDR vial 2_1_004.fcs · 170718 01099 50 nM MTG + 15 nM MTDR vial 2_2_005.fcs · 170718 01099 50 nM MTG + 15 nM MTDR vial 2_3_006.fcs |
