Detecting Apoptosis with VitaBright

Cell Vitality Analysis in 1 Minute

The cellular content of thiols such as glutathione (GSH) decreases as a specific response to apoptosis. To measure this, we developed two thiol-reactive fluorescent probes, VitaBright-43™ and VitaBright-48™, which can be used to detect apoptosis using the Vitality Assay for the NucleoCounter® NC-3000™.

Use VitaBright-43™ to detect early to mid-stage apoptotic events, and VitaBright-48™ for mid-stage to late apoptotic events. With the NucleoCounter® NC-3000™, your vitality analysis takes only one minute and is very easy to perform. You can run up to eight samples at a time.

Detect Early and Late Apoptotic Events

Free thiols play many important roles in cell biology. The predominant cellular oxidant is reduced glutathione (GSH), which protects against oxidative damage. The oxidation status of GSH largely determines the thiol-disulfide status (GSSG) and, hence, the cellular redox potential1. The concentration of GSH has been found to decrease upon induction of apoptosis due to GSH extrusion, also when inducing apoptosis using non-oxidative apoptogenic agents2-5.

VitaBright-43™ and VitaBright-48™ are cell-permeable maleimide derivatives that react with thiol groups on proteins resulting in thioester-coupled fluorescent products6,7. By using multicolor cytometry, it becomes clear that upon induction of apoptosis, a less intense VitaBright-43™ staining correlates with the timing of phosphatidylserine externalization and an increased Caspase 3/7 activity2. This makes VitaBright-43™ an ideal probe to detect early to mid-stage apoptotic events.

Cells stain less intensely for VitaBright-48™ during the later stages of apoptosis and this is inversely correlated to staining for the apoptotic marker Annexin V, and after the loss of mitochondrial potential as measured using a JC-1 stain7. Thus, VitaBright-48™ is an ideal marker for mid to late-stage apoptosis.

The Principle of VitaBright Staining and Analysis

When VitaBright is added to the cell culture, it crosses the cell membrane and immediately reacts with intracellular thiols, forming a blue-fluorescent compound. The intensity of the blue-fluorescence correlates directly with the cellular concentration of GSH. Moreover, the level of GSH and hence the level of free thiols decreases when apoptosis is induced. Therefore, measuring the level of free thiols can be used to quantify apoptosis.

 

VitaBright provides a very rapid, easy, and reliable way of assaying apoptosis by using either flow or image cytometry. Since no incubation or washing steps are required, these dyes help to preserve fragile apoptotic cells which are 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 (PI).

Vitality Analysis of Jurkat and WeHi-S cells

This figure (left) depicting the multiplex assay shows that VitaBright-43™ staining correlates well with phosphatidylserine externalization and Caspase 3/7 activity in Jurkat and Wehi-S cells. As apoptosis progresses, Annexin V CF-647 and NucView 488 signals increase while the VitaBright-43™ signal decreases in a subset of the cell population.

Panel A shows Jurkat cells treated with 5 µM camptothecin (CPT) for 0, 2 and 4 hours, respectively. Cells were stained with VitaBright-43™ (blue), Annexin V CF-647 (red) and SYTOX green (green) and analyzed using image cytometry with the NucleoCounter® NC-3000™. The scatter plots show VitaBright-43™ versus Annexin V CF-647 intensities. Non-viable cells were gated out based on SYTOX green uptake. The far-right panel shows an image of the CPT-treated sample.

Panel B shows WeHi-S cells treated with 10 ng/µl TNF-α for 0, 2 and 4 hours, respectively. Cells were stained with VitaBright-43™ (blue), NucView 488 (green) and PI (red) and analyzed using image cytometry with the NC-3000™. The scatter plots show VitaBright-43™ versus NucView 488 intensities. Non-viable cells were gated out based on PI uptake. The far-right panel shows an image of the TNF-α treated sample.

References

  1. SM Deneke and BL Fanburg: Regulation of cellular glutathione. Am J Physiol. 1989; 257(4 Pt 1):L163-73.
  2. L Ghibelli, S Coppola, G Rotilio et al.: Non-oxidative loss of glutathione in apoptosis via GSH extrusion. Biochem Biophys Res Commun. 1995; 216(1):313-20.
  3. AF Slater, CS Nobel, E Maellaro et al.: Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis. Biochem J. 1995; 306 (Pt 3):771-8.
  4. DJ v/d Dobbelsteen, CS Nobel, J Schlegel et al.: Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J Biol Chem. 1996; 271(26):15420-7.
  5. A Macho, T Hirsch, I Marzo et al.: Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. Immunol. 1997; 158(10):4612-9.
  6. ME Skindersoe, M Rohde and S Kjaerulff: A novel and rapid apoptosis assay based on thiol redox status. Cytometry A. 2012; 81(5):430-6.
  7. ME Skindersoe and S Kjaerulff: Comparison of three thiol probes for determination of apoptosis-related changes in cellular redox status. Cytometry A. 2014; 85(2):179-87.

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