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42239
CellSimple™ Mitochondrial Membrane Potential Assay Kit (I)
Assay Kit

CellSimple Mitochondrial Membrane Potential Assay Kit (I) #42239

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CellSimple cell-based analysis of live Jurkat cells untreated (left panel) or CCCP-treated (50 μM, 37ºC, 15 min; right panel) and labeled with JC-1 (2 μM, 30 min) using the CellSimple Mitochondrial Membrane Potential Assay Kit (I). Data was collected in both red (561 nm LP) and green (525/45 nm) channels and analyzed on the Open Flow Cytometry application. Note the marked decrease in mean fluorescence intensity in the red channel upon CCCP treatment. Instrument screen shots are shown.
Inquiry Info.# 42239
Product Includes Quantity (with Count) Solution Color
JC-1 3 x 15 µg
CCCP 1 x 100 µl
Phosphate Buffered Saline (PBS-20X) 9808 1 x 25 ml

Protocol

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Protocol for CellSimple™ Mitochondrial Membrane Potential (II) Assay #45898 and Mitochondrial Membrane Potential (II) Assay Protocol #13296

A. Instrumentation:

The CellSimple™ Mitochondrial Membrane Potential (II) Assay is specially designed for use with the CellSimple™ Cell Analyzer. However, either kit may be used with a flow cytometer or plate reader capable of providing excitation between 550 nm and 580 µM and detecting fluorescent emission at approximately 580 nm.

B. Kit components:

  • TMRE
  • CCCP
  • Phosphate Buffered Saline (PBS-20X)

C. Additional reagents needed, but not supplied.

  • DMSO
  • Reverse osmosis/deionized (RO/DI) water or equivalent

D. Reagent preparation

  1. 1X PBS: To prepare 500 ml 1X PBS add 25 ml PBS-20X to 475 ml RO/DI water, mix. Note: For flow cytometry application, adding 0.5% BSA to 1X PBS buffer may help to prevent cell loss.
  2. TMRE Stock Solution: Add 55 µl DMSO to each vial of TMRE to make a 1 mM stock solution. Aliquot if desired and store at -20°C. Each vial includes enough TMRE for 100 CellSimple™ tests, 50 flow cytometry assays (10 µl/assay) or five 96-well plates (0.1 µl/well), assays. 2 µM is used in this protocol. A titration between 0.1 to 10 µM is recommended to determine the optimal concentration for different cell lines.
  3. TMRE Labeling Solution: Dilute TMRE Stock Solution 1:500 with full cell culture medium to make 2 µM TMRE Labeling Solution.
  4. CCCP: Allow the 50 mM CCCP solution to equilibrate to room temperature before use

E. Protocol for suspension cells

  1. Suspend cells in warm media or PBS at 1 x 106 cell/ml. Prepare 1 ml aliquots; each 1 ml cell aliquot is one assay point. Make sure there are enough cells for your experiment. For example, if one compound is going to be assayed at three different concentrations, a total of 4 x 1 ml samples will be needed (this includes a positive control).
  2. Add test compound(s) to sample tubes at desired concentration and incubate cells for desired time. For best results, a compound titration and incubation time course can help to determine the best assay conditions. To prepare the positive control (mitochondrial membrane potential loss), add 1 µl of 50 mM CCCP to the control tube for a 50 µM final concentration; incubate cells at 37°C for 15 min.
  3. Add 100 µl of the TMRE Labeling Solution to each sample (200 nM final concentration) and incubate cells in the incubator (37°C and 5% CO2) for 15 to 30 min.
  4. Centrifuge sample at 300 x g for 5 min then remove the supernatant.
  5. Wash cells once with 1 ml warm 1X PBS, repeat step 4.
  6. Resuspend cells into 1000 µl warm 1X PBS.
  7. Analyze sample using an appropriate instrument. For analysis using the CellSimple™ Cell Analyzer use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Please see the CellSimple™ user guide for more details about using the Open Flow Application. If samples are to be analyzed on a plate reader, transfer 100 µl/ cell suspension/well to a black 96-well plate with a clear bottom and read using the following settings: excitation at approximately 550 nm and emission at approximately 580 nm.

F. Protocol for adherent cells

  1. Plate cells in a 96 well plate in warm culture medium place in incubator overnight to allow cells to attach to the plate. A typical cell number is between 1 x 104 and 5 x 104 cells/well. A cell number titration may be necessary for optimal results.
  2. Aspirate media from the plate and add test compounds in growth medium or 1X PBS to the plate at 100 l/well and incubate cells for desired time. Compound titration and incubation time course can help determine the best assay conditions. For a positive control (mitochondrial membrane potential loss), add CCCP to the control wells at 50 µM final concentration and incubate cells at 37°C for 15 min. For example, add 1 µl of 50 mM stock CCCP to 100 µl medium to make 500 µM CCCP; then add 10 µl of this 500 µM CCCP to each well containing 100 µl medium to get final concentration of 50 µM.
  3. Add 10 µl of TMRE Labeling Solution to each well to get a final concentration of 200 nM and place plate in an incubator (37°C and 5% CO2) for 20 min. Note: 200 nM TMRE is recommended in this protocol. For best results, a titration of TMRE is recommended.
  4. Aspirate the solution from the plate.
  5. Wash plate 3 times with warm 1X PBS and then add 100 µl/well 1X PBS to the plate.
  6. Analyze sample with an appropriate instrument. For analysis using the CellSimple™ Cell Analyzer, use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Please see the CellSimple™ user guide for more details about using the Open Flow Cytometry Application. If samples are to be analyzed on a plate reader, transfer 100 µl cell suspension/well to a black 96-well plate with a clear bottom and read using the following settings: excitation at approximately 550 nm and emission at approximately 580 nm.

posted June 2016

revised November 2016

Protocol Id: 1151

Product Description

CellSimple Mitochondrial Membrane Potential Assay Kit (I) is a fluorescent assay designed for use with the CellSimple Cell Analyzer. It detects the mitochondrial membrane potential in living cells. The kit includes the cationic dye JC-1 and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). JC-1 is a cell membrane permeable, fluorescent dye with green emission (~520 nm). When JC-1 accumulates in intact mitochondria, the dye forms aggregates that lead to orange-red fluorescence (~590 nm). The mean fluorescence intensity (MFI) of the orange-red emission can be used as an indicator for mitochondrial membrane potential.

Specificity / Sensitivity

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology:

All Species Expected

Background

The CellSimple Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.
Mitochondria function as the main cellular powerhouse and play important roles in other processes, such as steroid metabolism, calcium homeostasis, apoptosis, and cellular proliferation. Mitochondrial membrane potential is a key indicator of mitochondrial function and cell health (1,2). The dissipation of mitochondrial membrane potential is considered an early indicator of apoptosis (3).

JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) is a cell membrane permeable, cationic dye. In normal cells, JC-1 concentrates in mitochondria to form aggregates in response to high membrane potential. Decreased mitochondrial membrane potential results in dispersal of mostly monomeric JC-1 throughout the cell. When excited at 490 nm, JC-1 monomers emit a green fluorescence with a maximum at ~520 nm. Aggregates of JC-1 emit an orange-red fluorescence with a maximum at ~590 nm. Therefore, the fluorescence intensity of the orange-red emission and the ratio of orange-red fluorescence to green fluorescence can be used to measure mitochondrial membrane potential and serve as an indicator of overall cell health (4).

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