研究目的
Investigating the existence of lipid microdomains in the plasma membrane of living cells using fluorescent dyes and kinetic analysis.
研究成果
The study demonstrates a new kinetic approach to detect plasma membrane heterogeneity in living cells using fluorescent dyes and MEM decomposition, consistently revealing two exponential components attributed to liquid-ordered and disordered lipid phases. This provides evidence for the coexistence of lipid microdomains.
研究不足
The technique may have limitations in detecting more than two lipid phases or in different cell types. Further work is needed to comprehend the perspectives and limitations of this technique.
1：Experimental Design and Method Selection:
The study uses kinetic measurements of fluorescent dye binding and diffusion in cell membranes, decomposed using the Maximum Entropy Method (MEM) to identify exponential components. Three types of experiments were conducted: spectroscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP).
2：Sample Selection and Data Sources:
Human histiocytic lymphoma Jurkat cells and human epithelial adenocarcinoma HeLa cells were cultured in RPMI 1640 media with fetal bovine serum and antibiotics. Fluorescent probes NR12S, bNR10S, bQ10S, and F2N12SM were used.
3：List of Experimental Equipment and Materials:
Equipment includes PTI QuantaMaster 40 spectrofluorometer, BeckmanCoulter EpicsXL flow cytometer, ZEISS LSM 780-FCS confocal microscope, Matlab and ImageJ software. Materials include cell culture media, fluorescent dyes, and Ringer's solution.
4：Experimental Procedures and Operational Workflow:
For spectroscopy and flow cytometry, cells were stained with dyes, and fluorescence kinetics were measured. For FRAP, cells were bleached and recovery was monitored. Data were analyzed using MEM for exponential decomposition.
5：Data Analysis Methods:
MEM was used for decomposing time-dependent fluorescence signals into exponential components. Statistical analysis was performed using ANOVA in Matlab.
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ZEISS LSM 780-FCS
LSM 780-FCS
ZEISS
Used for confocal microscopy and FRAP experiments.
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PTI QuantaMaster 40
QuantaMaster 40
PTI
Used for fluorescence spectroscopy measurements, including steady-state spectra and kinetics.
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BeckmanCoulter EpicsXL
EpicsXL
BeckmanCoulter
Used for flow cytometry measurements of cell fluorescence.
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Matlab
R2015a 8.5.0.197613
MathWorks
Used for data analysis, including MEM decomposition and statistical tests.
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ImageJ
1.49 m
Used for image analysis in microscopy experiments.
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MEMfit
Software for Maximum Entropy Method decomposition of exponential components.
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