UNIT7.32 Uncompensated Polychromatic Analysis
of Mitochondrial Membrane Potential
Using JC-1and Multilar Excitation
volcanoSara De Biasi,1Lara Gibellini,1and Andrea Cossarizza1
1Department of Surgery,Medicine,Dentistry and Morphological Sciences,University of
Modena and Reggio Emilia,Modena,Italy
The lipophilic cation JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-
benzimidazolyl carbocyanine iodide)has been ud for more than20years
as a specific dye for measuring mitochondrial membrane potential( m).In
this unit,we revi our original protocol(that made u of a single488nm
lar for the detection of monomers and aggregates,and where compensation
was an important step)to u dual-lar excitation.Moreover,thanks to
revisions
recently developed multilar instruments and novel probes for surface and
intracellular markers,JC-1can be utilized by polychromaticflow cytometry
to simultaneously detect,without any compensation betweenfluorescences,
m along with other biological parameters,such as apoptosis and the
production of reactive oxygen species.C 2015by John Wiley&Sons,Inc.
Keywords:apoptosis r mitochondrial membrane potential r JC-1r polychro-
maticflow cytometry r Annexin V r CellRox
How to cite this article:
De Biasi,S.,Gibellini,L.,and Cossarizza,A.2015.Uncompensated
Polychromatic Analysis of Mitochondrial Membrane Potential
Using JC-1and Multilar Excitation.Curr.Protoc.Cytom.
empty的反义词72:7.32.1-7.32.11.
doi:10.0732s72
INTRODUCTION
The dissipation of the mitochondrial transmembrane potential( m)constitutes an
early and irreversible step in the cascade of events that,in veral cell types,can lead to
programmed cell death(apoptosis)(Galluzzi et al.,2012).
accompany
Several probes are available to measure m byflow cytometry,but some of them have
a low specificity for this organelle;conflicting data in the literature about the role of
m dissipation during the apoptotic process could be,at least in part,ascribed to this
lack of specificity.
After excitation with a blue lar at488nm,thefluorescent dye5,5ʹ,6,6ʹ-tetrachloro-
1,1ʹ,3,3ʹ-tetraethyl-benzimidazolyl carbocyanine iodide(JC-1),a lipophilic cation ex-
isting in a monomeric form,emits in the green region.However,in mitochondria
that have a high m,JC-1forms so called J-aggregates,described almost80years
ago(Jelley,1936),and undergoes a reversible change influorescence emission from
green to orange.Using commonflow cytometers equipped with such lars,for v-
eral years mitochondria have been studied by detecting the two emissions of JC-1
by the normalfilters prent in FL1(for monomers)and FL2(for aggregates)(Cos-
sarizza et al.,1993;Cossarizza et al.,1995;Polla et al.,1996;Cossarizza et al.,
1997;
Salvioli et al.,2000;Cossarizza et al.,2002;Lugli et al.,2007;Troiano et al.,2007; Gibellini et al.,2012;Abu et al.,2014;Marringa et al.,2014;also e older version Current Protocols in Cytometry7.32.1-7.32.11,April2015
Published online April2015in Wiley Online ).
doi:10.0732s72
Copyright C 2015John Wiley&Sons,Inc.Nucleic Acid Analysis
7.32.1 Supplement72
of this unit at /doi/10.0732s41/full).
Measurements using this dye provide information on changes in m(typically,a
decrea in m caus a relevant shift from orange to greenfluorescence emis-
sion),as well as on total mitochondrial content(bad on the intensity of the green
fluorescence emission).A number of studies have since shown the superiority of
JC-1over other dyes—e.g.,rhodamine123(R123)or3,3ʹ-dihexyloxadicarbocyanine
iodide[DiOC6(3)]—that were ud for the same purpo,and demonstrated that
JC-1is also unaffected by changes in plasma membrane potential(Salvioli et al.,
1997;Lugli et al.,2007;Troiano et al.,2007;also e older version of this unit at
/doi/10.0732s41/full).
This unit discuss a new method to detect JC-1(e Basic Protocol1),bad upon the
u of two lars,one to excite JC-1monomers(by the canonical488-nm lar line),
and the other to excite JC-1aggregates(by a yellow lar emitting at561nm).The
typical excitation by the blue lar excites JC-1with high efficiency,but sometimes
requires significant compensation between FL1and FL2.In contrast,yellow lar allows
a better resolution,and thus a clearer visualization of monomers and aggregates without
compensation(Perelman et al.,2012).For this reason,we have revid our basic JC-1
protocol using the two different lars quoted above.
Furthermore,we have recently developed another polychromaticflow cytometric assay
(e Basic Protocol2)utilizing JC-1and other probes for the simultaneous detection of m,reactive oxygen species(ROS,by CellRox DeepRed),and apoptosis(by Annexin V,detecting the exposure of phosphatidylrine on the plasma membrane).This protocol
can be applied when the simultaneous analysis of multiple parameters during apoptosis
is ,in investigating the role of certain proteins on cell phenotype or when
testing the cytotoxicity of compounds of pharmacological interest.
CAUTION:For the protection of laboratory personnel from potential infectious agents (e.g.,hepatitis and HIV),handle human samples using disposable gloves in a biological safety cabinet.
CAUTION:All probes described in this unit are potentially hazardous(e manufacturers’MSDSs),and urs should wear gloves during the staining procedures.
BASIC PROTOCOL1BASIC DETERMINATION OF MITOCHONDRIAL MEMBRANE POTENTIAL USING JC-1:DUAL-LASER EXCITATION OF THE DYE
A VOIDS COMPENSATION ISSUES
This protocol is intended for cells such as peripheral blood mononuclear cells(PBMCs)
or cell lines such as RKO,HL60,MCF7,and U937.Other cell types may also be stained
using minor adjustments to the steps described below.
Typically,by using a488-nm blue lar,it can be obrved that cells with high m
(that form JC-1aggregates)emit orangefluorescence(atß590nm);tho with low m(containing JC-1in its monomeric form)emit greenfluorescence(atß520nm) (Cossarizza et al.,1993).Recently it has been demonstrated that alternative excitation
wavelengths can facilitate the detection of m,and,most importantly,u of two
wavelengths avoids the need for compensation.Indeed,the excitation wavelength561nm
(i.e.,yellow lar)is above the emission spectra of JC-1monomers,and lectively
excites J-aggregates;hence there is no need to compensate green and orangefluorescence
(Perelman et al.,2012).Thus,we have adapted our original protocol(that made u of
a single488-nm lar,and where compensation was an important step)to an instrument
equipped with a blue and a yellow lar(like the Attune NxT,from Life Technologies).
Analysis of
Mitochondrial
Membrane
Potential Using
JC-1and
Multilar
Excitation
7.32.2
Supplement72Current Protocols in Cytometry
Materials
Experimental samples:human peripheral blood lymphocytes or monocytes,or
human tumor cell ,RKO,HL60,U937,MCF7);here we u RKO
cells,which derive from a colon carcinoma and grow adherent to the plasticflask Complete RPMI culture medium,1ml per sample
1M valinomycin[dissolve valinomycin(mol.wt.1111.32;Sigma-Aldrich)in
dimethylformamide(DMF)and store in a glass container up to6months at4°C]
or1mM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone(FCCP;
Sigma Aldrich)
2.5mg/ml JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolyl-
carbocyanine iodide):prepare by dissolving JC-1(Life Technologies,Thermo
Fisher Scientific)in dimethylformamide(DMF);store in a glass container up to
2years at–20°C,protected from light
Phosphate-buffered saline(PBS)
3.5-ml,55×12–mm plastic tubes(Sarstedt,or equivalent)
Centrifuge(Minifuge RF;Heraeus),or equivalent
Flow cytometer equipped with a488-nm blue lar and with a561-nm yellow lar,
<,Attune NxT(Life Technologies)
Additional reagents and equipment for counting(APPENDIX3A)and culturing
(APPENDIX3B)mammalian cells
Prepare cells
1.Count a sample of the experimental cells of interest(APPENDIX3A).
This protocol can be ud to stain either cells growing in suspension or adherent cells
after they have been relead from the plate by trypsinization(APPENDIX3B)and counted
账簿登记(APPENDIX3A).
2.Collect at least2×105cells from the experimental samples in55×12–mm tubes
by centrifuging5min at300×g,room temperature.Collect the same number of
cells to u for a positive control.
3.Decant and discard the medium and resuspend the cell pellet in1ml fresh complete
RPMI culture medium.
4.For obtaining a so-called“positive control,”i.e.,a sample where all cells have de-
polarized mitochondria,prepare one sample of cells treated with valinomycin(final
concentration0.1μM)or with carbonyl cyanide p-(trifluoromethoxy)phenylhydra-
zone(FCCP,final concentration250nM).Incubate10min or45min,respectively,
at37°C.
Drugs such as the K+ionophore valinomycin or the proton translocator FCCP are able
to collap theΔΨm.
Note that to avoid problems related to intracellular drug metabolism,in some instances
valinomycin is preferred over FCCP or ClCCP(and is also less expensive).
Stain with JC-1
5.Add1μl of2.5mg/ml JC-1fluorescent probe(2.5μg/mlfinal concentration)to the
experimental and positive control cells and shake the cell suspension until the dye
is well disperd and gives a uniform red-violet color.
providesJC-1tends to form aggregates when added to normal aqueous medium.To avoid this,
add the probe while gently vortexing.
6.Incubate the samples10min in the dark,37°C.Nucleic Acid
Analysis
7.32.3 Current Protocols in Cytometry Supplement72
Figure 7.32.1Changes in JC-1fluorescence after mitochondrial membrane depolarization in RKO cells treated with valinomycin,as described in Basic Protocol 1.Samples were acquired using 488-nm lar only (A ),or with dual-lar excitation (B ).Control cells (CTR)were stained with 2.5μg/ml JC-1.Note the shift to the bottom and to the right of cells with mitochondria depolarized by treatment with 100nM valinomycin.Right panel shows the merging of the left and center panels.Green-orange compensation was ß4%and orange-green compensation was ß10%;compensation was required to better visualize monomers and aggregates.All reagents must be at room temperature and carefully checked for pH (7.4)when ud,becau ΔΨm is very nsitive to alterations of the conditions.The staining procedure must be carried out away from direct inten light,and incubation must be in the dark becau of the light nsitivity of JC-1.7.Wash the cells by centrifuging 5min at 300×g ,room temperature,discarding the supernatant,and resuspending the cells in 1ml PBS for analysis on cytometer.Set up flow cytometer 8.Detect JC-1fluorescence of the experimental and positive control samples using a classical green band-pass filter centered at 525/50nm for monomers detection (channel of blue lar)and a classical greenish orange band-pass filter centered at 585/42nm (usually tho for a channel collecting fluorescence signals deriving from the excitation with the blue or the yellow lar).The most common flow cytometers are typically equipped with only a 488-nm argon or solid-state lar;no special requirements are needed to analyze ΔΨm .The gain of photomu
ltipliers (PMTs)obviously depends on the cytometer ud,but generally JC-1does not require any substantial increa in PMT amplification;green-orange compensation can be ß4%and orange-green compensation ß10%.However,note that no compensation is needed if a blue and a yellow lar are ud to detect monomers and aggregates,respectively.See Figure 7.32.1for a typical example of JC-1staining of control (CTR)RKO cells,and of RKO cells treated with valinomycin.Detection was performed by using a single blue lar (A)or using blue and yellow lars (B).This treatment results in a relevant change in the fluorescence distribution:cells with depolarized mitochondria can be easily identified as tho going from the center of the plot to the lower right quadrant.Analysis of
Mitochondrial Membrane Potential Using初中语文知识点总结
JC-1and
Multilar Excitation
7.32.4
Supplement 72Current Protocols in Cytometry
9.On the basis of the lar ud,adjust the voltage of the respective PMTs to obtain the bivariate gre
en versus orange distributions similar to tho shown in Figure
7.32.1A and B,and then record the control sample.U the same PMT ttings for the subquent samples.
Analyze JC-1stained experimental samples
10.Acquire fluorescence data for experimental samples in listmode,using a log scale
for the fluorescence channels.
Cells with high ΔΨm are tho forming J-aggregates;thus,they show high orange fluorescence.On the other hand,cells with low ΔΨm are tho in which JC-1maintains (or re-acquires)its monomeric form,and thus show green fluorescence.Once mitochondria are depolarized,JC-1monomers redistribute in other membranous compartments with lower ΔΨ.As a conquence,the green fluorescence intensity of depolarized cells is a little bit higher than that of polarized ones simply becau of the prence of a higher amount of JC-1monomers inside the cell.
11.Recommended for samples with heterogeneous cell populations:Set a gate on the
population of interest,then proceed with adjustment of PMTs,as well as compen-sation if a 488-nm lar is ud.Dual-lar excitation of the dye does not require compensation.
When the sample contains a heterogeneous cell population,it is possible to e different fluorescence patterns due to different autofluorescences and the variable content in terms of membranes and mitochondria of cell subpopulations.This is the ca for peripheral blood mononuclear cells (PBMCs),lymphocytes,and monocytes,the first being smaller and having fewer mitochondria than the latter.Accordingly,the fluorescence pattern of JC-1for such a sample shows at least two distinct peaks,one corresponding to lympho-cytes,and the cond,brighter in both green and orange,corresponding to monocytes.It is thus recommended to first t a gate on the population of interest,then proceed with adjustment of PMTs and compensation.
BASIC PROTOCOL 2ANALYSIS OF
M ,APOPTOSIS,AND REACTIVE OXYGEN SPECIES
CONTENT BY 4-LASER POLYCHROMATIC FLOW CYTOMETRY
This protocol allows the analysis of m along with the detection of early apoptotic cells,and the quanti
fication of the amount of reactive oxygen species in the cells of interest.It has been developed taking into account the possibility of simultaneously using four lars (by using an Attune NxT from Life Technologies)and avoiding any compensation among dyes.
幸运星英文
Fine analysis of the apoptotic process requires the detection of multiple cell functions at the same time,and it could be highly informative to reveal whether cells with different m also differ with respect to other parameters.This assay is recommended when studying compounds that can have differential effects on the cell populations of interest.This protocol us three different probes:JC-1(for m ),annexin V conjugated with Pacific Blue (for detecting the exposure of phosphatidylrine on the plasma membrane,a well known phenomenon which identifies early apoptotic cells),and CellRox Deep Red (for measuring ROS production).CellRox is a cytoplasmic cell-permeable non-fluorescent (or very weakly fluorescent)reagent which,in a reduced state and upon oxidation,exhibits a strong fluorogenic signal.CellRox Deep Red can be excited by a 638-nm lar,and emits at ß665nm.For complete information regarding the probes described here,e Internet Resources at the end of this unit.
Annexins are a family of soluble proteins (13different isoforms)with four to eight repeats of a 75–amino acid connsus quence relevant for Ca 2+binding.They are involved in membrane transport,
regulation of protein kina C,formation of ion channels,
Nucleic Acid Analysis 7.32.5Current Protocols in Cytometry Supplement 72
endocytosis,exocytosis,and membrane-cytoskeleton interactions.Annexin V binds with peculiar specificity to phosphatidylrine residues,which are precociously expod on the external leaflet of the plasma membrane during apoptosis (Lizarbe et al.,2013).Thus,when cells are annexin V positive,they have entered into an early pha of apoptosis.The annexin V–Pacific Blue conjugate is violet excitable,making it ideal for instruments with a lar at 405nm,and for multicolor experiments that include green-or red-fluorescent dyes.The Pacific Blue-conjugated annexin V emits at ß455nm after excitation by a violet light source.Before starting with sample analysis,running samples stained with single fluorochromes (e steps below)is suggested to properly t up fluorescence levels.Note that also in this ca there are no compensation requirements.Materials Cells in culture (ATCC):in suspension or adherent in 24-well tissue culture plate (as in Basic Protocol 1,we u RKO cells derived from human colon carcinoma Complete RPMI culture medium Phosphate-buffered saline (PBS)CellRox Deep Red Reagent (Life Technologies)2.5mg/ml JC-1(5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide);prepare by dissolving JC-1(Life Technologies,Thermo Fisher Scientific)in dimethylformamide (DMF);store in a glass container up to 2years at –20°C,protec
cablecar
ted from light Annexin V binding buffer (e recipe)Pacific Blue-conjugated annexin V (Life Technologies,Thermo Fisher Scientific):store at 4°C,protected from light 3.5ml,55×12–mm plastic tubes (Sarstedt,or equivalent)Centrifuge (Minifuge RF;Heraeus),or equivalent.Attune NxT cytometer or equivalent cytometer equipped with four light sources for excitation at 405nm (violet lar,for Annexin V),488and 561nm (blue and yellow lars,for JC-1),and 638nm (red lar,for CellRox)and filters for collecting fluorescence emissions at 455/40(for annexin V),520/20(for JC-1monomers),585/42(JC-1aggregates),and 660/40(CellRox)Additional reagents and equipment for counting (APPENDIX 3A )and culturing (APPENDIX 3B )mammalian cells and detaching adherent cells using trypsin (e APPENDIX 3B )Prepare cells 1.Count a sample of the cells in culture (e APPENDIX 3A ).For cells in suspension 2a.Collect at least 3×105cells from experimental samples by centrifuging 5min at 300×g ,room temperature.Collect the same number of cells to u for a positive control.3a.Decant and discard the medium and bring the total volume up to 1ml with prewarmed RPMI culture medium.For adherent cells 2b.Decant and discard the growth medium.3b.Add 1ml prewarmed culture medium (RPMI or similar)to the cells in the plate.Analysis of Mitochondrial Membrane Potential Using
吉林大学珠海分校JC-1and Multilar Excitation
7.32.6
Supplement 72Current Protocols in Cytometry
