Journal Club—Multiparameter Detection of Early Apoptosis Markers Without Compensation
Uncompensated polychromatic analysis of mitochondrial
membrane potential using JC-1 and multilaser excitation
Disruption of the mitochondrial membrane potential has been shown to accompany cell stress and early apoptosis. De Biasi and coworkers recently described a new version of their original flow cytometry protocol to measure mitochondrial membrane potential using the lipophilic cation JC-1. The JC-1 dye reversibly accumulates in mitochondrial membranes in a potential-dependent manner: at low membrane potential, JC-1 exhibits green fluorescence (~530 nm) and exists as a monomer; at higher potential, it forms red-fluorescent (~590 nm) “J-aggregates” in the mitochondrial membrane. The ratio of green to red fluorescence emission of JC-1 is thus proportional to the degree of mitochondrial membrane depolarization. Because early apoptotic cells exhibit disrupted mitochondrial membrane potential, JC-1 can be used as an early marker of apoptosis, alone or in combination with other apoptosis probes.
In the authors’ original JC-1 detection protocol, a flow cytometer equipped with only a single 488 nm (blue) laser was used for the analysis, requiring compensation between the two detection channels for JC-1 monomer and J-aggregate emission. The updated method described in this article employs the Attune™ NxT Acoustic Focusing Cytometer, an instrument available with up to four lasers (blue, red, violet, and yellow lasers). The use of multiple lasers for excitation minimizes the need for compensation, allowing for a simpler and more efficient detection protocol. Multilaser excitation also permitted the authors to develop a second protocol for the simultaneous detection of mitochondrial membrane potential changes and additional apoptosis markers, including the externalization of phosphatidylserine and the formation of reactive oxygen species.
The updated JC-1 detection protocol described by De Biasi et al. makes use of two lasers for JC-1 detection: the blue (488 nm) laser to excite JC-1 monomers and the yellow (561 nm) laser to excite J-aggregates. As shown in Figure 1, U937 cells were treated with valinomycin, a K+ ionophore that depolarizes the mitochondrial membrane, and then stained with JC-1. The treated cells are shifted to the bottom-right of the plot, representing an increase in JC-1 monomers. When single-laser excitation is used without any compensation, the J-aggregate and JC-1 monomer emissions cannot be easily separated in the plot of red vs. green fluorescence (Figure 1A); with dual-laser excitation, however, no compensation is required to distinguish the fluorescence emission of the two forms of JC-1 (Figure 1B).
For simultaneous detection of multiple markers of apoptosis, the authors used two additional probes: Pacific Blue™ annexin V (to detect exposed phosphatidylserine residues with the violet laser) and CellROX™ Deep Red Reagent (to detect reactive oxygen species with the red laser). Using four lasers simultaneously on the Attune™ NxT cytometer without compensation, they demonstrated the detection of all three apoptosis markers (Figure 2). They suggest that these protocols can be adapted for multiparameter detection of apoptosis using a variety of fluorescent cell function probes.
Figure 1. Uncompensated multilaser analysis of JC-1 fluorescence after mitochondrial membrane depolarization in U937 cells. Control U937 (human leukemic monocyte lymphoma) cells (CTR) were stained with 2.5 μg/mL JC-1; experimental U937 cells were treated with 1 μM valinomycin (VAL) for 15 min to depolarize mitochondria prior to JC-1 staining. Samples were acquired without compensation on the Attune™ NxT cytometer using (A) only a 488 nm laser for excitation and 530/30 nm and 574/26 nm bandpass (BP) filters for detection of JC-1 monomers and J-aggregates, respectively; or (B) both the 488 nm and 561 nm lasers for excitation and 530/30 nm and 585/16 nm BP filters for detection of JC-1 monomers and J-aggregates, respectively. Small insets show individual CTR and VAL plots; large plots show merged CTR and VAL data in order to visualize the JC-1 monomer and J-aggregate signals together. Data provided by Sara De Biasi, Lara Gibellini, and Andrea Cossarizza, University of Modena and Reggio Emilia, Italy.
Figure 2. Uncompensated multilaser analysis of apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) production in RKO cells. RKO colon cancer cells were cultured for 24 hr in the (A) absence or (B) presence of 5 μM CDDO (2-cyano-3,12-dioxo-oleana-1, 9(11)-dien- 28-oic acid, methyl ester), a Nrf2 activator that can inhibit cell proliferation and induce differentiation and apoptosis. Cells were stained with Pacific Blue™ annexin V, JC-1, and CellROX™ Deep Red Reagent, and data were collected using the 4-laser Attune™ NxT Acoustic Focusing Cytometer without compensation. Viable and apoptotic cells were identified by negative and positive Pacific Blue™ annexin V staining, respectively (405 nm laser, 440/50 nm BP filter); mitochondrial membrane potential was analyzed with JC-1 (488 nm and 561 nm lasers with filters described in Figure 1); reactive oxygen species (ROS) production was detected using CellROX™ Deep Red Reagent (637 nm laser, 670/40 nm BP filter). Reprinted from De Biasi S, Gibellini L, Cossarizza A (2015) Curr Protoc Cytom 7.32.1–7.32.11, with permission from Current Protocols in Cytometry.
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