Early in apoptosis, phosphatidylserine (PS) translocates from the inner (cytoplasmic) to the outer leaflet of the plasma membrane, an event which has historically been considered as irreversible. Polarity Sensitive Indicator of Viability & Apoptosis (pSIVA) is an Annexin XII based probe that is conjugated to the polarity sensitive dye, IANBD*, and specifically detects PS externalization. This novel probe exhibits enhanced properties compared to classical Annexin V based probes and is used in both flow cytometry and microscopy.
pSIVA’s membrane bound dependent fluorescence and reversible binding properties are a technological leap for detecting PS exposure.
PS-Dependent Fluorescence: In solution, pSIVA-IANBD does not emit any significant fluorescence. However, when pSIVA-IANBD binds to PS, the IANBD labels are exposed to the nonpolor lipid environment of the plasma membrane resulting in a ‘switching on’ of the IANBD fluorescence signal.
Reversible Binding: Following homeostatic or rescuable cell death events, PS flips back to the cytoplasmic face. This leads to the release of pSIVA-IANBD from the cell membrane and the loss of PS-dependent fluorescence.
Figure 1. pSIVA-IANBD: A polarity sensitive, reversible-binding probe for detecting exposed PS residues in the lipid membrane bilayer. Propidium Iodide (PI) is a vital dye used to identify late apoptosis or necrosis which are both associated with loss of membrane integrity.
BENEFITS OF pSIVA OVER ANNEXIN V
pSIVA assays are extremely straightforward because the probe is added directly to cells or tissues, incubated and analyzed. Unbound pSIVA lacks background fluorescence in contrast to Annexin V-based probes which require a wash step to remove the unbound probe and its inherent fluorescence. Besides improving wash-free assay sensitivity, pSIVA enables close monitoring of PS exposure throughout cell death/cell survival processes.
Detect PS exposure
No wash step required
Detects irreversible and transient PS
In vivo imaging
Table 1. Comparison of pSIVA to Annexin-V based probes. pSIVA is a novel generation Annexin probe for detecting PS exposure, with more features than Annexin V.
Figure 2. Comparison of pSIVA-IANBD and Annexin V-FITC in Flow Cytometry. The staining patterns were similar for the two probes:
Untreated: Primarily negative indicating healthy cells. The small positive population represents the basal level of cell death in the culture.
Staurosporine (1 h): Large positive population indicating active apoptosis. Staining in the ‘negative’ population gate has spread out, indicating movement towards cell death.
Staurosporine (3 h): Nearly the entire population is positive, indicating massive cell death.