研究目的
To describe a method to measure the level of reactive oxygen species (ROS) in Caenorhabditis elegans using CM-H2DCFDA to assess mitochondrial functional integrity.
研究成果
The protocol provides a reliable method for measuring ROS levels in C. elegans using CM-H2DCFDA, which helps in assessing mitochondrial health and oxidative stress, with applications in studying neurodegenerative diseases.
研究不足
Worm lysates for ROS measurement cannot be frozen for future use. The protocol may be confounded by autofluorescence in live animals, but this is bypassed by using lysates. FUDR treatment might affect mitochondrial function, and starved animals should not be used.
1：Experimental Design and Method Selection:
The protocol involves using CM-H2DCFDA, a cell-permeant chloromethyl derivative of a reduced form of fluorescein, which is oxidized by ROS to produce a fluorescent signal. This method is chosen to bypass autofluorescence issues in live imaging by using worm lysates.
2：Sample Selection and Data Sources:
C. elegans strains are obtained from the Caenorhabditis Genetics Center (CGC) and grown on NGM plates with E. coli OP50 as food source. Animals are synchronized at L1 stage by starvation and grown to day 1 adults for analysis.
3：List of Experimental Equipment and Materials:
Includes Petri dishes, centrifuge tubes, pipettes, sonicator, centrifuge, autoclave, microscope, microplate reader, and various reagents such as CM-H2DCFDA, BCA protein assay kit, and buffers like M9 and PBS.
4：Experimental Procedures and Operational Workflow:
Steps include growth and synchronization of nematodes, preparation of worm lysates by freezing, sonication, and centrifugation, incubation with CM-H2DCFDA dye, and fluorescence measurement using a microplate reader. Data are analyzed with normalization and statistical tests.
5：Data Analysis Methods:
Fluorescence intensity is measured at excitation 485 nm and emission 535 nm. Data are normalized by subtracting no-worm control values and further to wild type, then analyzed using one-way ANOVA with Bonferroni post-test in GraphPad Prism.
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Conical tubes
430829, 430791
Corning
Used for sample storage and centrifugation.
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Chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate
C6827
Thermo Fisher Scientific, Invitrogen?
Dye used to measure ROS levels by fluorescence.
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Microscope
SteREO Discovery.V8
ZEISS
Used for observing nematodes.
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Petri dishes
2900, 2901
Kord-Valmark Labware Products
Used for growing C. elegans on NGM plates.
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Centrifuge tubes
6285
Globe Scientific
Used for holding and centrifuging samples.
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Coverslip
1404-10
Globe Scientific
Used in microscopy.
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Pasteur pipettes
6-72050-900
Krackeler Scientific
Used for transferring liquids.
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Vacuum filtration
6722-5001
GE Healthcare, Whatman
Used for sterilizing solutions.
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Micro Centrifuge tube
229443
CELLTREAT Scientific
Used for small volume samples.
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C. elegans strains and OP50
Caenorhabditis Genetics Center (CGC)
Biological samples used in the experiment.
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BCA protein assay
23227
Pierce, Thermo Fisher Scientific, Thermo Scientific?
Used to measure protein concentration in lysates.
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Polybead Polystyrene microspheres
00876-15
Polysciences
Possibly used as standards or in procedures (not specified in detail).
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Agarose
A20090-500.0
RPI
Used in media preparation.
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Single channel pipettes
PR-10, PR-20, PR-200, PR-1000
Rainin
Used for precise liquid handling.
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Multichannel pipettes
Fisherbrand? Finnpipette? II, 21377830
Fisher Scientific
Used for high-throughput liquid handling.
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Sonic Dismembrator
Sonic Dismembrator Model 100
Fisher Scientific
Used to sonicate worm samples for lysate preparation.
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Centrifuge
IEC Centra CL2
Thermo Fischer Scientific, Thermo Scientific?
Used for centrifuging samples.
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Centrifuge
5415 D
Eppendorf
Used for centrifuging small samples.
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Autoclave
433/533HC-E
Getinge, Castle Steam Sterilizer
Used for sterilizing equipment and media.
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Microplate Reader
FlexStation? 3
Molecular Devices
Used to measure fluorescence intensity.
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