BD Pharmingen™ MitoStatus Reagents
Assessing Mitochondrial Membrane Potential on the
BD Accuri™ C6 Flow Cytometer
Features
Cationic, lipophilic dyes for assessment of mitochondrial
membrane potential in a single fluorescence channel
Detection of mitochondrial depolarization, a marker
of apoptosis, autophagy, senescence, and other cellular
processes
Expanded choice and flexibility in multicolor panel
design
B
2,200
Camptothecin vs DMSO
B02 CAM
Gate: P1
Characteristic
MitoStatus TMRE
MitoStatus Red
Excitation peak
549 nm
622 nm
Emission peak
574 nm
648 nm
488 nm (blue)
640 nm (red)
Detector
1,000
Count
1,000
Count
Laser
Equivalent
fluorochromes*
FL2
PE
FL4
APC
Alexa Fluor® 647
*Do not use these fluorochromes in the same tube with the corresponding MitoStatus dye.
Table 1. Fluorescence characteristics of MitoStatus dyes on the BD Accuri C6.
0
0
MitoStatus TMRE
A
2,200
FCCP vs DMSO
10 1
10 2
10 3
10 4
10 5
10 6
10 7.2
10 1
10 2
10 3
10 4
10 5
10 6
10 7.2
0
Count
1,000
D
In cells undergoing apoptosis, oxidative stress, necrosis, and other
cellular processes, the mitochondrial membrane can become
depolarized. For example, in apoptosis, pro-apoptotic Bcl-2 family
proteins cause mitochondrial outer membrane permeabilization
(MOMP), resulting in the release of cytochrome c and the
subsequent activation of caspase-9 and the apoptotic cascade.
MOMP often correlates with the loss of Δψm, which can be
detected using Δψm-sensitive dyes. Figure 1 shows that MitoStatus
TMRE and MitoStatus Red clearly detect reduced Δψm in cells
treated with FCCP (a mitochondrial uncoupler) or camptothecin
(which induces apoptosis).
C02 CAM
Gate: P1
1,000
C03 FCCP
Gate: P1
2,200
FL2 MitoStatus TMRE-A
0
Count
C
2,200
FL2 MitoStatus TMRE-A
MitoStatus Red
On the BD Accuri™ C6 flow cytometer, BD Pharmingen™
MitoStatus TMRE dye is excited by the blue laser and
BD Pharmingen™ MitoStatus Red dye by the red laser, allowing
flexibility in multicolor panel design. Table 1 shows their
fluorescence characteristics.
Tested for compatibility with the BD Accuri C6
B03 FCCP
Gate: P1
BD Pharmingen™ MitoStatus reagents can be used to assess a
cell’s inner mitochondrial membrane potential (Δψm), which is
central to the study of apoptosis, autophagy, senescence, and
other cellular processes. These cationic, lipophilic dyes accumulate
within the mitochondria of healthy cells, but not within
mitochondria that have lost Δψm due to induction of apoptosis
or treatment with a mitochondrial uncoupler. Typically, the
fluorescence intensity of healthy cells will be approximately 10-fold
higher than cells whose mitochondrial membranes are depolarized.
10 1
10 2
10 3
10 4
10 5
10 6
10 7.2
10 1
FL4 MitoStatus Red-A
DMSO (control)
10 2
10 3
10 4
10 5
10 6
10 7.2
FL4 MitoStatus Red-A
Camptothecin
FCCP
Figure 1. Detecting mitochondrial depolarization with MitoStatus reagents
Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 0.025%
DMSO vehicle control for 5 hours (red), 5 μM camptothecin for 5 hours to
induce apoptosis (blue), or 50 μM FCCP mitochondrial uncoupler for 20 minutes
(green). Cells were then stained with 100 nM of BD Pharmingen MitoStatus TMRE
(upper plots) or MitoStatus Red (lower plots) for 15 minutes at 37°C in media,
washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and
acquired and analyzed on the BD Accuri C6 using BD Accuri™ C6 software.
Results: A, C. Compared to the vehicle-treated controls, Jurkat cells treated with
FCCP mitochondrial uncoupler show a decrease in fluorescence intensity due to
depolarization of mitochondria. B, D. Camptothecin-treated cells show a mix of
polarized and depolarized populations as they progress through apoptosis.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
The MitoStatus dyes are similar to BD Pharmingen™ Annexin V
dyes (Cat. Nos. 556570 and 559763), in that both can be used
to detect apoptosis. While the MitoStatus dyes measure Δψm,
Annexin V measures phosphatidylserine exposure. However, the
MitoStatus dyes can be used in experiments where Annexin V is
unsuitable, such as those that involve cells sensitive to calcium or
certain adherent cells. Also, as Figure 2 shows, Δψm is often a
slightly earlier sign of apoptosis than phosphatidylserine exposure.
Finally, the MitoStatus dyes can detect not only apoptosis but also
autophagy, senescence, and other processes that are associated
with Δψm changes.
BD Pharmingen™ MitoStatus Reagents
10 5
10 4
10 5
10 6
10 2
10 7.2
10 1
10 2
Q7-UL
2.2%
Q7-UR
7.4%
Dead
10 4
10 5
10 6
10 1
10 2
10 3
Q8-LR
83.2%
83.2%
10 4
10 5
10 6
10 7.2
B02 CAM
Gate: Cells
Q10-UL
9.6%
Dead
Q10-UR
0.0%
10 5
FL2 PI-A
10 4
10 3
FL2 PI-A
10 7.2
10 7.2
F
10 6
10 7.2
B02 CAM
Gate: Cells
10 6
10 3
Apoptotic
Q8-LL
4.0%
FL4 MitoStatus Red-A
10 5
Transitional
Q2-LL
6.0%
10 2
10 4
Apoptotic
/Dead
Q2-LR
69.4%
10 1
10 7.2
Q2-UR
0.4%
10 5
10 6
E
10 3
10 2
FL4 MitoStatus Red-A
10 1
Camptothecin
10 3
Live
FL1 Annexin V FITC-A
B02 CAM
Gate: Cells
Q2-UL
24.2%
Live
10 2
10 4
FL2 PI-A
10 1
10 1
10 2
10 7.2
Q8-UR
0.1%
Live
Q7-LL
26.4%
10 1
FL1 Annexin V FITC-A
10 2
Apoptotic
Q7-LR
64.0%
10 3
10 4
10 5
10 6
10 7.2
FL1 Annexin V FITC-A
10 4
10 6
Q8-UL
12.8%
Dead
Live
10 3
10 5
Apoptotic
Q4-LR
4.6%
10 2
10 4
Live
Q4-LL
82.5%
10 1
10 3
10 4
10 5
10 2
10 1
10 1
FL2 PI-A
10 5
10 4
Apoptotic
/Dead
Q3-LR
10.3%
Transitional
Q3-LL
6.1%
FL1 Annexin V FITC-A
D
Q4-UR
10.3%
Dead
10 6
Q4-UL
2.6%
A02 DMSO
Gate: Cells
10 3
10 7.2
Q3-UR
0.9%
10 6
Q3-UL
82.7%
Live
C
A02 DMSO
Gate: Cells
10 3
10 7.2
B
A02 DMSO
Gate: Cells
10 3
10 2
FL4 MitoStatus Red-A
10 1
DMSO (control)
10 6
A
10 7.2
Easy to use, simple to maintain, and affordable, the BD Accuri
C6 personal flow cytometer is equipped with a blue laser, a red
laser, two light scatter detectors, and four fluorescence detectors.
A compact design, fixed alignment, and pre-optimized detector
settings result in a system that is simple to use. A nonpressurized
fluidics system enables kinetic measurements in real time. For
walkaway convenience, the optional BD CSampler™ accessory
(Cat. No. 653124) offers automated sampling from 24-tube racks
or multiwell plates.
In detecting Δψm status, the MitoStatus reagents are also similar
to JC-1, used in the BD™ MitoScreen Kit (Cat. No. 551302).
However, JC-1 is a ratiometric dye that requires two fluorescence
channels, while the MitoStatus dyes require only one. JC-1 may
still be preferable for cell types whose mitochondrial number can
vary widely, since healthy cells with few mitochondria will bind
little MitoStatus dye, and thus may appear falsely depolarized.
Apoptotic
Q10-LL
66.0%
10 1
10 2
10 3
Q10-LR
24.4%
10 4
10 5
10 6
10 7.2
FL4 MitoStatus Red-A
Figure 2. Detecting apoptosis with MitoStatus Red and Annexin V
Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 0.025% DMSO vehicle control (upper plots) or 5 μM camptothecin (lower plots) for 5 hours to induce
apoptosis. Cells were then stained with 100 nM of BD Pharmingen MitoStatus Red for 15 minutes at 37°C in media, washed twice with BD Pharmingen Stain Buffer (FBS)
(Cat. No. 554656), and stained with BD Pharmingen™ Annexin V FITC and propidium iodide (PI) according to the BD Pharmingen™ Annexin V FITC Apoptosis Detection
Kit staining protocol (Cat. No. 556570). Cells were acquired and analyzed on the BD Accuri C6 using BD Accuri C6 software. Results: A, D. Co-staining with Annexin V
FITC and MitoStatus Red revealed two major populations: live cells with polarized mitochondria and no phosphatidylserine exposure (UL), and apoptotic or dead cells with
depolarized mitochondria and exposed phosphatidylserine (LR). A transitional population with depolarized mitochondria but no phosphatidylserine exposure (LL) reveals the
loss of mitochondrial membrane potential to be a slightly earlier marker of apoptosis in these cells. B, C, E, F. Co-staining with either Annexin V FITC (B, E) or MitoStatus
Red (C, F) and PI can detect live, apoptotic, and dead populations. Both co-stains reveal an increase in the number of apoptotic populations after camptothecin treatment,
with similar percentages of live, apoptotic, and dead cells in the corresponding gates. (Quadrant gates are set based on the live cell population and adjusted along the PI
axis to account for apoptotic cells being semi-permeable to PI.) Use of MitoStatus TMRE would yield similar results (data not shown).
Ordering Information
Description
Quantity
Cat.No.
BD Pharmingen™ MitoStatus TMRE
25 mg
564696
BD Pharmingen™ MitoStatus Red
100 µg
564697
Related Products
Description
Cat.No.
BD™ MitoScreen (JC-1) Kit
551302
BD Pharmingen™ Annexin V Apoptosis
Detection Kit
556570 (FITC)
559763 (PE)
BD Pharmingen™ Stain Buffer (FBS)
554656
BD Accuri™ C6 Flow Cytometer System
653118
BD CSampler™ Automated Sampling System
653124
Class1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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