Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm*
Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. 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. Our proprietary Apopxin™ PS sensor used in this kit is small molecule-based PS sensor. The green fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense green fluorescence at ~520 nm. The kit is optimized to be used with a flow cytometer equipped with Violet Laser. It is particularly suitable for multicolor flow cytometric analysis of cells. In coupling with its large Stokes Shift, its highly enhanced affinity to PS makes this kit 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.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds (200 µL/sample)
- Add Apopxin™ Violet 500 assay solution
- Incubate at room temperature for 30 - 60 mintues
- Analyze cells using flow cytometer with 525/40 nm filter (AmCyan channel) or fluorescence microscope with Violet filter set
Important notes
Thaw 100X Propidium Iodide (Component C) at room temperature before starting the experiment.
SAMPLE EXPERIMENTAL PROTOCOL
Prepare and incubate cells with Apopxin™ Violet 500:
- Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
- Centrifuge the cells to get 1 - 5×105 cells/tube.
- Resuspend cells in 200 µL of Assay Buffer (Component B).
- Add 2 µL of Apopxin™ Violet 500 (Component A) into the cells.
- Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells for necrosis cells.
- Incubate at room temperature for 30 to 60 minutes, protected from light.
- Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.
- Monitor the fluorescence intensity using a flow cytometer with 525/40 nm filter (AmCyan channel) or a fluorescence microscope with Violet filter set.
Analyze by using a flow cytometer:
- Quantify Apopxin™ Violet 500 binding using a flow cytometer with 525/40 nm filter (AmCyan channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells. Note: Apopxin™ binding flow cytometric analysis on adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.
Analyze by using a fluorescence microscope:
- Pipette the cell suspension after incubation, rinse 1 - 2 times with Assay Buffer, and then resuspend the cells with Assay Buffer.
- Add the cells on a glass slide that is covered with a glass cover-slip. Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip. After incubation with Apopxin™ Violet 500, rinse 1 - 2 times with Assay Buffer, and add Assay Buffer back to the cover-slip. Invert cover-slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after the incubation with Apopxin™ Violet 500 and visualized under a microscope.
- Analyze the apoptotic cells with Apopxin™ Violet 500 under a fluorescence microscope with Violet filter. Measure the cell viability using TRITC filter when propidium iodide is added into the cells. The blue staining on the plasma membrane indicates the Apopxin™ Violet 500 binding to PS on cell surface.
Citations
View all 2 citations: Citation Explorer
STE20-Type Kinases MST3 and MST4 Act Non-Redundantly to Promote the Progression of Hepatocellular Carcinoma
Authors: Caputo, Mara and Xia, Ying and Anand, Sumit Kumar and Cansby, Emmelie and Andersson, Emma and Marschall, Hanns-Ulrich and K{\"o}nigsrainer, Alfred and Peter, Andreas and Mahlapuu, Margit
Journal: (2023)
Authors: Caputo, Mara and Xia, Ying and Anand, Sumit Kumar and Cansby, Emmelie and Andersson, Emma and Marschall, Hanns-Ulrich and K{\"o}nigsrainer, Alfred and Peter, Andreas and Mahlapuu, Margit
Journal: (2023)
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
Authors: Su, Yifan and Tian, Hua and Wei, Lijiang and Fu, Guohui and Sun, Ting
Journal: Acta Biochimica et Biophysica Sinica (2018): 658--665
References
View all 135 references: Citation Explorer
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
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
Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
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Authors: Wang CY, Lin YS, Su WC, Chen CL, Lin CF.
Journal: Mol Biol Cell (2009): 4153
Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation
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Journal: Toxicol Appl Pharmacol (2009): 144
Authors: Bae ON, Lim KM, Noh JY, Chung SM, Kim SH, Chung JH.
Journal: Toxicol Appl Pharmacol (2009): 144
Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry
Authors: Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM.
Journal: Exp Cell Res (2009): 1706
Authors: Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM.
Journal: Exp Cell Res (2009): 1706
Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53
Authors: Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ.
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Authors: Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ.
Journal: Cancer Cell Int (2009): 16
Page updated on December 1, 2024