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pSIVA™ Microscopy Kit

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1x105 HeLa cells were seeded on 4-well glass slides (sterile) and cultured in 0.5 ml RPMI medium supplemented with 2 mM CaCl2. Cells were treated with 1 µM of the apoptosis inducing agent staurosporine (Figures E, F, G, H) or left untreated (Figures A, B, C, D). pSIVA–IANBD + PI were added to the media according to kit instructions. Cells were imaged post treatment with the Zoe Fluorescent Cell Imager at 0 minutes, 60 minutes, 120 minutes and 270 minutes. Images A-H are merged images of the red and green fluorescence channels. Early apoptotic cells were stained with pSIVA-IANBD and are shown in green while late apoptotic/dead cells were stained with PI and are shown in red.

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pSIVA™ REAL-TIME Apoptosis Fluorescent Microscopy Kit

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pSIVA™ Microscopy Kit

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The process of apoptosis is undertaken in several stages defined by specific cellular morphologies. One of the earlier stages of apoptosis is a change of the plasma membrane’s phospholipid asymmetry. This rearrangement results in the translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane (in non-apoptotic cells PS is exclusively located to the inner plasma membrane). However, apoptosis is reversible until reaching a certain point in the pathway and until then PS exposure can be considered as a transient event. The event defining whether the cell can be rescued and continues living is the onset of mitochondrial outer membrane permeabilization (Chipuk et al. 2006). Prior to reaching this point, PS exposure may be transient as molecules can relocate back to the inner plasma membrane (a phenomenon known as “PS flipping”) (van der Mark et al. 2013).

The pSIVA (polarity-Sensitive Indicator of Viability & Apoptosis) probe is a biosensor conjugated to the green emitting IANBD dye (excitation maximum 488 nm, emission maximum 530 nm) and only fluoresces when bound to PS in the presence of Ca2+ (Kim et al. 2010a, 2010b). The method thereby allows the analysis of kinetic apoptosis events in real time by live cell imaging and immunofluorescence / immunocytochemistry. In contrast to other PS detection based assays (e.g. annexin V) the pSIVA Real-Time Apoptosis Fluorescent Microscopy Kit does not require washing steps as you can simply add the probe and start analyzing.

Reagents in the Kit
pSIVA-IANBD 200 μl
Propidium Iodide Staining Solution 500 μl
Regulatory
For research purposes only.
Guarantee
6 months from date of despatch.
Acknowledgements
pSIVA is a trademark of Novus Biologicals and is protected under patent no. 8.541.549.

Store at +4°C. DO NOT FREEZE.
This product should be stored undiluted. This product is photosensitive and should be protected from light.

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
Application Name Verified Min Dilution Max Dilution
Immunocytochemistry Refer to Instructions For Use
Immunofluorescence Refer to Instructions For Use
Live Cell Imaging Refer to Instructions For Use
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Instructions For Use
Prior to commencing the microscopy experiment, please ensure that your cell culture medium contains between 1-2 mM Ca2+. Ca2+ is essential for binding of the pSIVA-IANBD probe to exposed phosphatidylserine (Kim et al. 2010b). If Ca2+ levels are insufficient, supplement the culture medium with 2 mM Ca2+.

1. Seed cells into culture plates and allow cells to adhere.
2. Optional. After 24 hours exchange the culture medium for medium containing 2 mM Ca2+, if required.
3. Optional. Induce apoptosis by treating cells with apoptosis inducing agents such as staurosporine or camptothecin.
4. Add 10–20 μl/ml* of the pSIVA-IANBD probe to cells. Mix gently by moving culture plates
backwards and forwards and side to side to ensure even distribution of the probe. DO NOT PIPETTE TO MIX.
5. Optional. If distinction between apoptotic and necrotic/dead cells is desired, add between 5–10 μl/ml* of propidium iodide (PI) to cells. Mix gently by moving plates backwards and forwards and side to side to ensure even distribution of PI. DO NOT PIPETTE TO MIX.
6. Observe cells under microscope using the green fluorescence filter for pSIVA-IANBD (excitation maximum 488 nm, emission maximum 530 nm) and the red fluorescence filter for PI (excitation maximum 535 nm, emission maximum 617 nm) visualization.
* The stated pSIVA-IANBD and PI quantities are guidelines only and may have to be optimized.

pSIVA Real-time Apoptosis Fluorescent Microscopy Kit Instructions.

Description Product Code Applications Pack Size List Price Your Price Quantity
Annexin V:APC Assay Kit ANNEX200APC F 200 Tests
List Price Your Price
Description Annexin V:APC Assay Kit
Annexin V:Pe Assay Kit ANNEX200PE F 200 Tests
List Price Your Price
Description Annexin V:Pe Assay Kit
Annexin V:APC Assay Kit ANNEX50APC F 50 Tests
List Price Your Price
Description Annexin V:APC Assay Kit
Annexin V:Pe Assay Kit ANNEX50PE F 50 Tests
List Price Your Price
Description Annexin V:Pe Assay Kit

References for pSIVA™ Microscopy Kit

  1. Kim, Y.E. et al. (2010) (a) Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration.
    Nat Methods 7(1): 67–73.
  2. Kim, Y.E. et al. (2010) (b) Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis.
    Nat Protoc. 5(8): 1396-405.

Synonyms
Annexin 12
Annexin XII

APO004

154355 158313

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