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Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Orange Fluorescence Optimized for Microplate Readers*

OverviewpdfSDSpdfProtocol


This particular kit is designed to monitor cell apoptosis through measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed. This kit uses our proprietary orange fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has orange fluorescence upon binding to membrane PS. The stain has the spectral properties almost identical to those of Cy3® or Alexa Fluor® 555, making it convenient to be used for the common fluorescence instruments equipped with the light sources and filters for Cy3® or Alexa Fluor® 555 (Cy3® or Alexa Fluor® 555 are the trademarks of GE Healthcare and Invitrogen respectively). Due to its highly enhanced affinity to PS, this kit is more robust than the other commercial Annexin V-based apoptosis kits that are only used with either microscope or flow cytometry platform. This kit can be also used with a fluorescence microplate reader besides the microscope and flow cytometry platforms.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: Apopxin™ Orange (100X Stock Solution)1 vial (100 µL/vial)
Component B: Assay Buffer (4 °C)1 bottle (10 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  2. Add equal volume of Apopxin™ Orange working solution
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or fluorescence microscope with Cy3 filter

Important notes
Warm Assay Buffer (Component B) at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 10 μL of 100X Apopxin™ Orange (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Apopxin™ Orange working solution. Note: 100 μL of Apopxin™ Orange working solution is enough for one well. Prepare fresh before use.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells with test compounds by adding 10 µL/well (96-well plate) or 2.5 µL/well (384-well plate) of 10X test compound stock solution into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.

  2. Incubate the cell plate in a 5% CO2, 37°C incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Apopxin™ Orange working solution into each well.

  4. Incubate the cell plate at room temperature for at least 1 hour, protected from light.

  5. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).

  6. Monitor the fluorescence intensity with a a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or image cells using fluorescence microscope with Cy3® filter.

Citations


View all 4 citations: Citation Explorer
Integrin $\beta$3 inhibits hypoxia-induced apoptosis in cardiomyocytes
Authors: Su, Yifan and Tian, Hua and Wei, Lijiang and Fu, Guohui and Sun, Ting
Journal: Acta Biochimica et Biophysica Sinica (2018): 658--665
Tumor-selective mitochondrial network collapse induced by atmospheric gas plasma-activated medium.
Authors: Saito, Kosuke and Asai, Tomohiko and Fujiwara, Kyoko and Sahara, Junki and Koguchi, Haruhisa and Fukuda, Noboru and Suzuki-Karasaki, Miki and Soma, Masayoshi and Suzuki-Karasaki, Yoshihiro
Journal: Oncotarget (2016)
Physiological effects of the herbicide glyphosate on the cyanobacterium Microcystis aeruginosa
Authors: Wu, Liang and Qiu, Zhihao and Zhou, Ya and Du, Yuping and Liu, Chaonan and Ye, Jing and Hu, Xiaojun
Journal: Aquatic Toxicology (2016): 72--79
Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole--imidazole polyamide, which targets an E-box motif
Authors: Taniguchi, Masashi and Fujiwara, Kyoko and Nakai, Yuji and Ozaki, Toshinori and Koshikawa, Nobuko and Toshio, Kojima and Kataba, Motoaki and Oguni, Asako and Matsuda, Hiroyuki and Yoshida, Yukihiro and others, undefined
Journal: FEBS open bio (2014): 328--334

References


View all 92 references: Citation Explorer
Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 22512
Peptidic targeting of phosphatidylserine for the MRI detection of apoptosis in atherosclerotic plaques
Authors: Burtea C, Laurent S, Lancelot E, Ballet S, Murariu O, Rousseaux O, Port M, V and er Elst L, Corot C, Muller RN.
Journal: Mol Pharm (2009): 1903
Detection of apoptosis based on the interaction between annexin V and phosphatidylserine
Authors: Liu T, Zhu W, Yang X, Chen L, Yang R, Hua Z, Li G.
Journal: Anal Chem (2009): 2410
Evaluation of cell surface expression of phosphatidylserine in ovarian carcinoma effusions using the annexin-V/7-AAD assay: clinical relevance and comparison with other apoptosis parameters
Authors: Dong HP, Holth A, Kleinberg L, Ruud MG, Elstr and MB, Trope CG, Davidson B, Risberg B.
Journal: Am J Clin Pathol (2009): 756
Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 6918
Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation
Authors: Bae ON, Lim KM, Noh JY, Chung SM, Kim SH, Chung JH.
Journal: Toxicol Appl Pharmacol (2009): 144
Discovery of a phosphatidylserine-recognizing peptide and its utility in molecular imaging of tumour apoptosis
Authors: Thapa N, Kim S, So IS, Lee BH, Kwon IC, Choi K, Kim IS.
Journal: J Cell Mol Med (2008): 1649
Caspase-dependent and -independent induction of phosphatidylserine externalization during apoptosis in human renal carcinoma Cak(1)-1 and A-498 cells
Authors: Lock EA, Reed CJ, Kinsey GR, Schnellmann RG.
Journal: Toxicology (2007): 79
Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis
Authors: Balasubramanian K, Mirnikjoo B, Schroit AJ.
Journal: J Biol Chem (2007): 18357
PET imaging of apoptosis with (64)Cu-labeled streptavidin following pretargeting of phosphatidylserine with biotinylated annexin-V
Authors: Cauchon N, Langlois R, Rousseau JA, Tessier G, Cadorette J, Lecomte R, Hunting DJ, Pavan RA, Zeisler SK, van Lier JE.
Journal: Eur J Nucl Med Mol Imaging (2007): 247