流式细胞仪测定线粒体膜电位的操作方法
JC-1 Mitochondrial Membrane
Potential Assay Kit
Item No. 10009172
TABLE OF CONTENTS
GENERAL INFORMATION
3 Materials Supplied4 Precautions
4 If You Have Problems4 Storage and Stability
4 Materials Needed but Not Supplied
INTRODUCTION 5 Background
5 About This Assay
PRE-ASSAY PREPARATION 6 Reagent Preparation
ASSAY PROTOCOL
7 Flow Cytometry
8 Fluorescence Microscopy9 Plate Reader
P ERFORMANCE CHARACTERISTICS 10 Representative Staining Results
RESOURCES 12 Troubleshooting
13 References14 Related Products
15 Warranty and Limitation of Remedy16 Notes
Kit will arrive packaged as a -20°C kit. For best results, remove components and store as
If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 975-3999. We cannot accept any returns without
prior authorization.
Precautions
Please read these instructions carefully before beginning this assay.For research use only. Not for human or diagnostic use.
Technical Service Contact Information
Phone: 888-526-5351 (USA and Canada only) or 734-975-3888Fax: 734-971-3641
Email: [email protected]: M-F 8:00 AM to 5:30 PM EST
In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box).
Storage and Stability
This kit will perform as specified if stored as directed in the page 3, and used before the expiration date indicated on the outside of the box.
Materials Supplied section, on Materials Needed But Not Supplied
1. Adjustable pipettes and a repeat pipettor2. A 6-, 12-, 24-, or 96-well plate for culturing cells
3. A flow cytometer, fluorescence microscope, or plate reader equipped with laser/
fluorescent filters capable of detecting the J-aggregates form of JC-1 using an excitation of 520-570 nm and an emission at 570-610 nm as well as the monomeric form of JC-1 at excitation and emission wavelengths of 485 and 535 nm, respectively.4. Distilled water
Apotosis is a cellular process involving a genetically programmed series of events leading to the death of a cell. During this process, several key events occur in mitochondria, including the release of caspase activators such as cytochrome c, changes in electron transport, and loss of mitochondrial transmembrane potential (parameter of mitochondrial function and has been used as an indicator of cell health.ΔΨm). 1 For this reason, ΔΨm is an important Variation of intensity of cells stained with cationic dyes such as rhodamine-123 (Rh123) and DiOCΔΨm has been previously studied by evaluating the changes in fluorescence However, data obtained by using these probes may not be reliable. For example, Rh123 6. 2 is relatively insensitive to depolarization of the plasma membrane and changes at ΔΨm changes and DiOC6 dye cannot distinguish between or pathological conditions when both events can take place.ΔΨm in several physiological 3cytofluorimetric, lipophilic cationic dye, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi- More recently, a new dazolylcarbocyanine iodide (Jcationic dyes in that it can selectively enter into mitochondria and reversibly change C-1), has been developed. JC-1 has advantages over other color from green to red as the membrane potential increases. In healthy cells with high mitochondrial intense red fluorescence. On the other hand, in apoptotic or unhealthy cells with low ΔΨm, JC-1 spontaneously forms complexes known as J-aggregates with JC-1 remains in the monomeric form, which shows only green fluorescence.
ΔΨm, About This Assay
Cayman’s Jbehavior of mitochondria in a variety of conditions, including apoptosis. The main advantage C-1 Mitochondrial Membrane Potential Assay Kit can be used to study the of this assay is that the changes in or red fluorescence can be both qualified and quantified by fluorescence microscopy, flow ΔΨm reflected by different forms of JC-1 as either green cytometry, or a fluorescence plate reader with appropriate filter sets.
NOTE: JC-1 is light sensitive. Do not expose to direct intense light.
Thaw the JC-1 Reagent at room temperature. Mix well. Tthis solution, we recommend that you make small aliquots and store them at -20°C.
o avoid repeated freeze/thawing of Reagent Preparation
1. Assay Buffer Preparation
Dissolve three Cell-Based Assay Buffer tablets (Item No. 10009322) in 300 ml of distilled water. This buffer should be stable for approximately one year at room temperature.
2. JC-1 Staining Solution Preparation
Thaw an aliquot of the JPrepare a staining solution by diluting the reagent 1:10 in the culture medium you C-1 Reagent (Item No. 10009908) at room temperature. are using for your cells. Mix well to make sure there are no particles or flakes in the solution.
NOTE: JC-1 Staining Solution is difficult to prepare due to its low solubility in aqueous medium and tendency to form particulates that are difficult to remove. Make sure JC-1 Reagent is coculture medium. Do not centrifuge the reagent.
mpletely thawed and warmed to roo m temperature before diluting it into
1. Culture cells in 6-, 12-, or 24-well plates at a density of 5 x 105 incubator overnight at 37°C. Tcells/ml in a CO2
(each sample should be run in duplicate or triplicate). Incubate the cells according to reat the cells with or without experimental compounds your normal protocol.2. Add 100 μl of the Jmedium to each well of the plate. For example, if you culture cells in 2 ml of culture C-1 Staining Solution (prepared on page 6) per ml of culture
medium in a 6-well plate, add 200 μl of the JC-1 Staining Solution into each well. Mix gently. Further dilution, such as adding only 50 μl of JC-1 Staining Solution to 1 ml of culture medium, may be used in cases when the staining is too intense.3. Incubate samples in a COis usually obtained after 15 minutes of incubation.2 incubator at 37°C for 15-30 minutes. Sufficient staining
4. Harvest cells from each well into a plastic tube fitted for the flow cytometer. The
samples can be directly analyzed in the culture medium.5. Analyze the samples immediately. Healthy cells with functional mitochondria contain
red JC-1 J-aggregates and are detectable in the FL2 channel. Apoptotic or unhealthy cells with collapsed mitochondria contain mainly green Jdetectable in the FL1 channel.C-1 monomers and are The following steps are optional:
6. Alternatively, centrifuge the samples obtained in step 4 (above) for five minutes at 400 x g at room temperature. Carefully aspirate the supernatant. Add 1 ml of Assay
Buffer to each tube and vortex to ensure that all cells are suspended.7. Centrifuge the samples for five minutes at 400 x g at room temperature. Carefully
aspirate the supernatant.8. Repeat steps 6-7 one more time.
9. Add 500 μl of Assay Buffer to each tube and vortex to ensure that all cells are
suspended in the assay solution.10. Analyze the samples immediately. Healthy cells with functional mitochondria contain
red JC-1 J-aggregates and are detectable in the FL2 channel. Apoptotic or unhealthy cells with collapsed mitochondria contain mainly green Jdetectable in the FL1 channel.
C-1 monomers and are
Fluorescence Microscopy
A 6-, 12-, 24-, or 96-well culture plate can be used for this method. We recommend that the cell density be ≤1 x 106 cells/ml. Optimal conditions will be dependent on the cell type.1.
Culture cells in 6-, 12-, 24-, or 96-well plates at a density of 5 x 105 incubator overnight at 37°C. Tcells/ml in a CO2 (each sample should be run in duplicate or triplicate). Incubate the cells according to reat the cells with or without experimental compounds your normal protocol.
2. Add 100 μl of the Jmedium to each well of the plate. For example, if you culture cells in 2 ml of culture C-1 Staining Solution (prepared on page 6) per ml of culture
medium in a 6-well plate, add 200 μl of the JC-1 Staining Solution into each well. Mix gently. Further dilution, such as adding only 50 μl of JC-1 Staining Solution to 1 ml of culture medium, may be used in cases when the staining is too intense.3. Incubate samples in a COis usually obtained after 15 minutes of incubation. The cells can be analyzed directly 2 incubator at 37°C for 15-30 minutes. Sufficient staining
in the culture medium since phenol red does not interfere with fluorescent staining. Healthy cells with mainly JC-1 J-aggregates can be detected with fluorescence settings usually designed to detect rhodamine (excitation/emission = 540/570 nm) or TRed (excitation/emission = 590/610 nm). Apoptotic or unhealthy cells with mainly exas JC-1 monomers can be detected with settings designed to detect FITC (excitation/emission = 485/535 nm).The following steps are optional:
4. Centrifuge the plate for five minutes at 400 x g at room temperature. Discard the
supernatant by careful aspiration.5. Add 2 ml, 1 ml, 500 μl, or 200 μl of Assay Buffer to each well of 6-, 12-, 24-, or
96-well plate respectively.6. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
aspirate the supernatant.7. Repeat steps 5-6 one more time.
8. Add 1 ml, 500 μl, 250 μl, or 100 μl of Assay Buffer to each well of 6-, 12-, 24-,
or 96-well plate, respectively. The cells are now ready for analysis by fluorescent microscopy and must be analyzed immediately. Healthy cells with mainly JJ rhodamine (excitation/emission = 540/570 nm) or Texas Red (excitation/emission -aggregates can be detected with fluorescence settings usually designed to detect C-1 = 590/610 nm). Apoptotic or unhealthy cells with mainly Jdetected with settings designed to detect FITC (excitation/emission = 485/535 nm).
C-1 monomers can be Plate Reader
A 96-well density be ≤1 x 10Black culture plate should be used for this method. We recommend that cell 6 cells/well. Optimal conditions will be dependent on the cell type.1. Culture cells in a 96-well black plate at a density of 5 x 104 - 5 x 105100 μl culture medium in a CO cells/well in
or without experimental compounds (each sample should be run in duplicate or 2 incubator overnight at 37°C. Treat the cells with triplicate). Incubate the cells according to your normal protocol.2.
Add 10 μl of the JC-1 Staining Solution (prepared above) to each well and mix gently. Further dilution, such as adding 5 μl of JC-1 Staining Solution to 100 μl of culture medium, may be used in cases where the staining is too intense.
3. Incubate the cells in a COis usually obtained after 15 minutes of incubation.2 incubator at 37°C for 15-30 minutes. Sufficient staining
4. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully
aspirate the supernatant.5. Add 200 μl of Assay Buffer to each well and centrifuge the plate for five minutes at
400 x g at room temperature. Carefully aspirate the supernatant.6. Repeat step 5 one more time.
7. Add 100 μl of Assay Buffer to each well. The cells are now ready for analysis by
a fluorescent plate reader. In healthy cells, Jstrong fluorescent intensity with excitation and emission at 560 nm and 595 nm, C-1 forms J-aggregates which display respectively. In apoptotic or unhealthy cells, Jstrong fluorescence intensity with excitation and emission at 485 nm and 535 nm, C-1 exists as monomers which show respectively. The ratio of fluorescent intensity of J-aggregates to fluorescent intensity of monomers can be used as an indicator of cell health.
Figure 1. Effect of staurosporine on mitochondrial potential in Jurkat cells.Jurkat cells were plated at a density of 5 x 104 treated with 2.5 μg/ml of staurosporine or vehicle for two hours in a CO cells/well. The next day, cells were 37°C. JC-1 Staining Solution was then added to the wells and staining was visualized 2 incubator at after a 15 minute incubation. strong J-aggregation (red). Panel A: untreated cells showing most of cells had cells stained green due to low Panel B:ΔΨm.
staurosporine treated cells showing a majority of
J-aggregates in control cells vs apoptotic cells
(induced by staurosporine)
87654321
Control
0.5 µg/ml2.5 µg/ml
Staurosporine Staurosporine
Figure 2. Measurement of JC-1 fluorescence in Jurkat cells in a 96-well plate format. Jurkat cells were plated at a density of 5 x 104cells were treated with staurosporine or vehicle for two hours in a CO cells/well. The next day, 37°C. JC-1 Staining Solution was then added to the wells and incubated at 37°C for 2 incubator at 15 minutes. The fluorescent intensity for both J-aggregates and monomeric forms of JC-1 was measured with a 96-well plate reader (J-aggregates: excitation/emission = 560/595 nm; JC-1 monomers: excitation/emission = 485/535 nm).
Troubleshooting
References
1. Green, D.R. and Reed, J.C. Mitochondria and apoptosis. 2. (1998).
Science 281, 1309-1312
Petit, Pfunction are early events of dexamethasone-induced thymocyte apoptosis. .X., Lecoeur, H., Zorn, E., et al. Alterations in mitochondrial structure and
J. Cell Biol. 3. 130(1)Salvioli, S., Ardizzoni, A., Franceschi, C., , 157-167 (1995).
123, is a reliable fluorescent probe to assess et al.for studies on mitochondrial functionality during apoptosis. ΔΨ JC-1, but not DiOC6(3) or rhodamine
changes in intact cells: Implications (1997).
FEBS Lett. 411, 77-82
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Warranty and Limitation of Remedy
Cayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material its delivery obligations with due care and skill. Thus, in no event will Cayman have any will meet our specifications at the time of delivery. Cayman will carry out obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees.
Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a of the purchase price, or at Cayman’s option, the refund material that does not meet our specifications.
replacement , at no cost to Buyer, of all Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material.
For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog.
NOTES
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06/04/2012, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed