细胞表面的PS外翻在1995年首次报道为细胞凋亡的显著特征，并被大量实验引用，长期以来，PS的外翻被认为是细胞凋亡中不可逆的行为，然而近年来文献报道PS的外翻是可逆的，不仅存在于凋亡细胞中，也存在于其他细胞形态中。pSIVA-IANBD技术的出现可以实时追踪PS的外翻，使科研工作者获得更多不同类型细胞死亡中的细胞膜极性变化信息。

pSIVA（Polarity Sensitive Indicator ofViability Apoptosis）凋亡检测技术是Novus2010年推出的一项新型技术，并于2013年发布专利。该技术使用的pSIVA是一种Ca2+依赖性蛋白，与磷酯酰丝氨酸（PS）有非常高的结合能力，其与IANBD极性染料偶联后形成pSIVA-IANBD复合物，这种复合物只有结合PS才会产生荧光。如下图所示：

而传统的AnnexinV检测技术，无论是否与PS结合，荧光始终存在，无法区分PS的瞬时与不可逆外翻，因此，相比较AnnexinV，pSIVA能够做到让细胞凋亡检测更精准。

NovuspSIVA凋亡检测试剂盒自上市以来，受到广大科研工作者的喜爱与热捧，该试剂盒操作相当简单，只需将pSIVA-IANBD或pSIVA-IANBD+PI直接加入细胞或组织，孵育后便可检测，无需洗涤！

[1]Neuronal deletion of caspase 8protects against brain injury in mouse models of controlledcortical impact and kainic acid-induced excitotox- icity. KrajewskaM, Z You, J Rong, C Kress, X Huang, J Yang, T Kyoda, R Leyva, SBanares, Y Hu, C-H Sze, MJ Whalen, L Salemena, R Hakem, BP Head, JCReed, S Krajewski. PLOS ONE 6(9): e24341.doi:10.1371/journal.pone.0024341 (2011). IF (mouse primary neuron cultures): Figs 2B,3.Cells were labeled with pSIVA-IANBD for 15 min, fixed and stainedwith a MAP2 antibody and DAPI. Cells double-stained with bothpSIVA-IANBD and MAP2 (neuronal marker) were identified asdegenerating neurons.

[2] Hostile takeover by Plasmodium:reorganization of parasite and host cell membranes during liverstage egress. Graewe S, KE Rankin, C Lehmann, C Deschermeier, LHecht, U Froehlke, RR Stanway, V Heussler. PLOS ONE 7(9): e1002224doi:10.1371/journal.ppat.1002224 (2011). IF (HepG2 cells infected with P. bergheiparasites), Fig 1

[3] Mitochondrial NDUFS3 regulatesthe ROS-mediated onset of metabolic switch in transformed cells.Suhane S, H Kanzaki, V Arumugaswami, R Murali, VK Ramanujan.Biology Open doi: 10.1242/bio.20133244 (2013).pSIVA-IANBD Flow Kit:Flow (Cell Surface): Fig 1(HEK293 cells). pSI- VA-IANBD was used to determine the basal levelof apoptosis in HEK cells.

[4] Monitoring apoptosis andneuronal degeneration by real-time detection of phosphatidylserineexternalization using a polarity-sensitive indica- tor of viabilityand apoptosis. Kim YE, J Chen, R Langen, JR Chan. Nature Protocols5:1396-1405 (2010b). Time lapsemicroscopy of neurons innormal survival conditions and after NGFdeprivation (Fig 2).

[5] A compact B model of huntingtintoxicity. Zhang CQ, Yeh T-l, A Leyva,LG Frank, J Miller, YE Kim, RLangen, S Finkbeiner, ML Amzel, CA Ross, MA Poirier. JBC286:8188-8196 (2011). ApSIVA-IANBD based cell suspen- sion toxicity assay was used todetermine cell viability in mouse Neu- ro2A (neuroblastoma)overexpressing huntingtin proteins (Fig 4).32 NBP2-29611 NBP2-29382 pSIVA TM Kit pSIVA TM Kit Manual

[6] Diurnal, localized exposure ofphosphatidylserine by rod outer seg-ment tips in wild-type but notItgb5-/- or Mfge8-/- mouse retina. RuggieroL, MP Connor, J Chen, RLangen, SC Finnemann. PNAS 109:8145-4148(2012). Live tissue imaging (mouse retina): Figs 4, 5. S4.pSIVA-IANBDwas added to dissected live mouse retina and shown tolabel the tips of photoreceptor outer segments (POS).The resultssuggested that phosphatidylserine (PS) exposure is specif-ic to thePOS surface. Furthermore, enhanced PS exposure preceded rodshedding and phagocytosis, suggesting that surface PS exposurepromotes these processes.

[7]Characterization of cell cyclemodifications induced by electric pulses in human cornealendothelium. Thi MH, Z He, N Campolmi, S Piselli, P Gain, M Peoc’H,JM Dumolllard, S Acquart, O Garraud, G Thuret. Acta Op-thalmologica 90:s249 (2012). Live tissue imaging (human corneaorgan cultures): pSIVA-IANBDwas used to assess cell death following gene electrotransfer intocorneal endothelial cells.

[8.]Cell death goes LIVE:Technological advances in real-time tracking of cell death. SkommerJ, Z Darzynkiewicz, D Wlodkowic. Cell Cycle 9:2330- 2341(2010). Live cell imaging(etoposide treated cell lines NGF-de-prived primary neuronalcultures): Discussion about tools for tracking cell deathreal-time.

[9]Annexin B12Cys101,Cys260-N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitro-benz-2-oxa-1,3-diazol-4-yl)ethylenediamine.Shan L In: Molecular Imaging and Contrast Agent Database (MICAD)[Internet]. Bethesda (MD): National Center for BiotechnologyInformation (US); 2004-2013.http://www.ncbi.nlm.nih.gov/books/NBK45028/ (2010)Live cell imaging: The story of pSIVAas a phosphatidylserine-targeted, real-time, apoptosis-detectingprobe.

[10] The change of cellularmembranes on apoptosis: Fluorescence detection. Demchenko AP. ExpOncol 34:263-268 (2012). Liveimaging: Discussion about pSIVA as an important advancement inannexin based methodology.

[11]Real-time flow cytometry forthe kinetic analysis of oncosis. Warnes G,S Martins. Cytometry PartA 79A:181-191 (2011). Liveimaging: Discussion about pSIVA as an assay for real-time detectionof apoptosis.

[12] Engineering apolarity-sensitive biosensor for time-lapse imaging of apoptoticprocessess and degeneration. Kim YE, J Chen, JR Chan, R Langen.Nature Methods 7:67-73 (2010a). Real-time live-cell imagingand time-lapsemicroscopy of apoptosis: Fig 2 (COS-7 cells), Fig 3 (rat33NBP2-29611 NBP2-29382 pSIVA TM KitpSIVA TM Kit Manualneuronal degeneration), Fig 4 (rat axonal degeneration), Fig 5(rescue of rat neuronal degeneration as visualized bypSIVA.

[13]Monitoring apoptosis andneuronal degeneraton by real-time detection of posphatidylserineexternalization using a polarity-sensitive indicator of viabilityand apoptosis. Kim YE, J Chen, R Langen, JR Chan. Nature Protocols5:1396-1405 (2010b). Time lapsemicroscopy of neurons innormal survival conditions and after NGFdeprivation (Fig 2).

[14] A compact beta model ofhuntingtin toxicity. Zhang CQ, Yeh T-I, A Leyva,LG Frank, J Miller,YE Kim, R Langen, S Finkbeiner, ML Amzel, CA Ross, MA Poirier. JBC286:8188-8196 (2011). ApSIVA-IANBD based cell suspen- sion toxicity assay was used todetermine cell viability in a mouse Neuro2A neuroblastoma cell lineoverexpressing huntingtin proteins (Fig 4).

[15]Hostile takeover by Plasmodium:reorganization of parasite and host cell membranes during liverstage egress. Graewe S, KE Rankin, CLehmann, C Deschermeier, LHecht, U Froehike, RR Stanway, V Heussler. PLOS ONE 7(9): e1002224doi:10.1371/journal.ppat.1002224 (2011). IF (HepG2 cells infected with P. bergheiparasites), Fig 1.

[16] Novel applications of pSIVA, anew polarity sensitive phosphatidylser-ine (PS) apoptosis/celldeath probe. Stein LS, PD Quintel, S Krajewski,JS Rosenberg, SKSingh. Cancer Res 72:237 (2012). Flow (Cell Surface):Jurkat (Figs 1-4). IF: Mouseprimary neuronal cultures (Figs 5-6).

[17] Diurnal photoreceptor outersegment shedding: contributions of the RPE. Finnemann SC, LRuggiero, MP Connor. Invest Opthalmol Vis Sci 53:4327 (2012). LiveImaging: mouse retina. pSIVA was used quantify the surface exposureof phosphotidylserine on the distal rod photore- ceptor outersegments.

[18] Flavopiridol synergizes withsorafenib to induce cytotoxicity and potentiate antitumorigenicactivity in EGFR/HER-2 and mutant RAS/RAF breast cancer modelsystems. Nagaria TS, JL Williams, C Leduc, JA Squire, PA Greer, WSangrar. Neoplasia 15:939-951 (2013). Flow cytometry (Cell surface): MDA-MB-231 (Fig 4A)and MDA-MB-468 (Fig 4B) adenocarcinoma cells.

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