研究目的
To present a detailed protocol for combining organelle-specific dyes with surface marker staining to quantify mitochondria and lysosomes in different T cell populations using multicolor flow cytometry.
研究成果
The protocol successfully enables quantification of mitochondrial and lysosomal contents in various T cell subsets, revealing fluctuations during development and activation. It overcomes limitations of traditional methods by allowing high-throughput analysis of rare and heterogeneous populations, with potential applications in studying metabolic changes in diseases like cancer and autoimmune disorders.
研究不足
The protocol requires accurate titration of dyes and careful control of temperature and light exposure to avoid reduced staining efficiency. It is limited by the flow cytometer's capability to handle multicolor fluorescence, with potential issues from spectral spillover when using many colors. Cell viability must be maintained, and immediate analysis is necessary due to dye vulnerability.
1：Experimental Design and Method Selection:
The protocol involves using multicolor flow cytometry to quantify organelles in T cells by combining organelle-specific dyes (MitoTracker Green and LysoTracker Green) with surface marker staining. This allows for high-throughput analysis of heterogeneous cell populations.
2：Sample Selection and Data Sources:
Mouse spleen and thymus tissues are harvested to obtain lymphocyte suspensions. Cells are processed to remove red blood cells and clumps.
3：List of Experimental Equipment and Materials:
Equipment includes flow cytometer, CO2 chamber for euthanasia, dissecting scissors, forceps, centrifuge, cell strainer (70 μm pore size), hemocytometer or automated cell counter, FACS tubes, incubator. Materials include FACS buffer (PBS with 2% FBS and 1 mM EDTA), ACK lysing buffer, complete RPMI medium, organelle-specific dyes (MitoTracker Green, LysoTracker Green), fluorescence-conjugated antibodies, propidium iodide (PI), 2.4G2 hybridoma supernatant.
4：4G2 hybridoma supernatant. Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Steps include tissue harvest and cell suspension preparation, staining with organelle-specific dyes at 37°C, surface marker staining on ice, sample acquisition on flow cytometer with gating and compensation, and data analysis using software like FlowJo.
5：Data Analysis Methods:
Data is analyzed by gating cell populations based on surface markers, calculating mean fluorescence intensity (MFI) for organelle staining, and comparing ΔMFI (staining minus FMO control) across populations.
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MitoTracker Green
Stains mitochondria in live cells for flow cytometry quantification.
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LysoTracker Green
Stains lysosomes in live cells for flow cytometry quantification.
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flow cytometer
Acquires and analyzes fluorescent signals from stained cells to quantify organelles and surface markers.
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cell strainer
70 μm pore size
Filters cell suspensions to remove clumps before flow cytometry analysis.
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FACS buffer
Buffer used for cell washing and staining in flow cytometry procedures.
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ACK lysing buffer
Lyses red blood cells in cell suspensions.
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complete RPMI medium
Culture medium for cell suspension and staining steps.
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propidium iodide
Stains dead cells to exclude them from flow cytometry analysis.
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2.4G2 hybridoma supernatant
Blocks Fc receptors to prevent non-specific antibody binding during surface marker staining.
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