VitaBright Stains – for Cytometric Profiling of Apoptosis
The level of thiols as gluthathione (GSH) decreases as a specific response to apoptosis. Therefore, the thiol-reactive fluorescent probes VitaBright -43 and VitaBright-48 can be used to detect apoptosis.
VitaBright-43™ and -48™ are cell-permeable maleimide derivatives that react with thiol groups on proteins to give thioester-coupled fluorescent products (Figure 1)1,2. Free thiols play a number of important roles in biology. An example is the predominant cellular oxidant, reduced glutathione (GSH), which protects against oxidative damage. GSH provides a large proportion of the reducing power available in the cell, and its oxidation status largely determines the thiol-disulfide status of the cell and, hence, the cellular redox potential3. The concentration of GSH has been found to decrease upon induction of apoptosis, also when using non-oxidative apoptogenic agents, due to extrusion of GSH4-7.
A) Schematic drawing showing the principle of intracellular detection of free thiols using VitaBright Stains. Upon addition to a cell culture VitaBright crosses the cell membrane and immediately reacts with intracellular thiols, forming a blue fluorescent compound.B) CHO cells stained with VitaBright-48™ (blue) and a nuclear marker (red).Interchange reactions links the cellular concentration of GSH directly to the level of other reduced thiols. Moreover, the level of GSH, and hence the level of thiols, decreases in response to induction of apoptosis. Thus, measuring the level of free thiols can be used to quantify apoptosis. The two thiol probes VitaBright-43™ and -48™ provide a very rapid, easy and reliable way of assaying apoptosis by either flow or image cytometry as no incubation or washing steps are required; this facilitates preservation of fragile apoptotic cells often lost during washing steps. To discriminate between necrotic and apoptotic cells VitaBright™ staining should be combined with an impermeable stain, such as propidium iodide. Using multicolor cytometry it has been demonstrated that VitaBright-43™ staining correlates well with phosphatidylserine externalization and Caspase 3/7 activity (figure 2)2.
Figure 2: Multiplex assays demonstrating that VitaBright-43™ staining correlates well with phosphatidylserine externalization and Caspase 3/7 activity; as apoptosis progresses Annexin V CF-647 and NucView 488 signals increase while VitaBright-43™ signal decreases.
A) Jurkat cells treated with 5 µM camptothecin (CPT) for 0, 2 and 4 hours, respectively. Cells were stained with VitaBright-43™, Annexin V CF-647 and SYTOX green and analysed by image cytometry using the NucleoCounter® NC-3000™ system. Plots show VitaBright-43™ intensity versus Annexin V CF-647 intensity. Nonviable cells were gated out based on SYTOX green uptake. Right panel shows an image of the CPT-treated sample.
B) WeHi-S cells treated with 10 ng/µl TNF-α for 0, 2 and 4 hours, respectively. Cells were stained with VitaBright-43™, NucView 488 and propidium iodide and analysed by image cytometry. Plots show VitaBright-43™ intensity versus NucView 488 intensity. Nonviable cells were gated out based on propidium iodide uptake. Right panel shows an image of the TNF-α treated sample.
VitaBright-48™ may also be used for measuring apoptosis. However, this probe detects changes in thiol level, and hence apoptosis, later than VitaBright-43™ 1,2. Table below summarizes some of the features.
Comparison of VitaBright-43™ and -48™
|Thiol Probe||Signal strength||Apoptosis||Staining kinetics||Organisms tested|
|Vitabright-43™||Bright||Early to mid-stage||Fast, no incubation||Mammalian and insect cells|
|Vitabright-48™||Very bright||Mid-stage to late||Fast, no incubation||Mammalian and insect cells|
VitaBright -43™ and -48™ now available in the WebShop.