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Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue 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 PS sensor has blue fluorescence upon binding to membrane PS. The PS stain used in the kit has the spectral properties similar to those of Pacific Blue® (Pacific Blue® is the trademark of Invitrogen). The blue fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense blue fluorescence at ~450 nm. The kit is optimized to be used with a flow cytometer equipped with a Violet Laser. It is particularly suitable for multicolor flow cytometric analysis of cells. This kit has been used for flow cytometric analysis of cells in multicolor applications in combination with fluorescent antibodies.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Apopxin™ Violet 450 assay solution
  3. Incubate at room temperature for 30 - 60 mintues
  4. Analyze cells using flow cytometer with 450/40 nm filter (Pacific Blue channel) or fluorescence microscope with Violet filter

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 450:

  1. Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  2. Centrifuge the cells to get 1 - 5×105 cells/tube.

  3. Resuspend cells in 200 µL of Assay Buffer (Component B).

  4. Add 2 µL of Apopxin™ Violet 450 (Component A) into the cells.

  5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells for necrosis cells.

  6. Incubate at room temperature for 30 to 60 minutes, protected from light.

  7. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.

  8. Monitor the fluorescence intensity using a flow cytometer with 450/40 nm filter (Pacific Blue channel) or a fluorescence microscope with Violet filter.

Analyze by using a flow cytometer:

  1. Quantify Apopxin™ Violet 450 binding using a flow cytometer with 450/40 nm filter (Pacific Blue 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:

  1. Pipette the cell suspension after incubation, rinse 1 - 2 times with Assay Buffer, and then resuspend the cells with Assay Buffer.

  2. 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 450, 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 450 and visualized under a microscope.

  3. Analyze the apoptotic cells with Apopxin™ Violet 450 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 450 binding to PS on cell surface.

Citations

View all 4 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)
The Growth, Apoptosis and Oxidative Stress in Microcystis viridis Exposed to Glyphosate
Authors: Ye, Jing and Huang, Chen and Qiu, Zhihao and Wu, Liang and Xu, Chao
Journal: Bulletin of Environmental Contamination and Toxicology (2019): 1--5
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
Integrin β3 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)

References

View all 135 references: Citation Explorer
Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
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
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
Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53
Authors: Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ.
Journal: Cancer Cell Int (2009): 16
Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 6918
Page updated on December 17, 2024

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Catalog Number22835
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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation405 nm laser
Emission450, 40 nm filter
Instrument specification(s)Pacific Blue channel

Fluorescence microscope

ExcitationViolet filter
EmissionViolet filter
Recommended plateBlack wall, clear bottom

Components

The detection of phosphatidylserine binding activity in Jurkat cells with Cell Meter™ Phosphatidylserine Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Apopxin™ Violet 450 for 30 minutes. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using violet laser.
The detection of phosphatidylserine binding activity in Jurkat cells with Cell Meter™ Phosphatidylserine Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Apopxin™ Violet 450 for 30 minutes. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using violet laser.
The detection of phosphatidylserine binding activity in Jurkat cells with Cell Meter™ Phosphatidylserine Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Apopxin™ Violet 450 for 30 minutes. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using violet laser.
Hypoxia inhibits cell proliferation and induces cell apoptosis. H9C2 cells were treated with CoCl2 (900 μM) or a hypoxic microenvironment (1% O2) for 24 h. Cell apoptosis was detected by flow cytometry. *P < 0.05, **P < 0.01. Cell Meter™ Phosphatidylserine Apoptosis Assay Kit (AAT Bioquest) was used to visualize apoptotic cells according to the manufacturer’s instructions. <b>Source:</b> Integrin β3 inhibits hypoxia-induced apoptosis in cardiomyocytes by Yifan Su, Hua Tian, Lijiang Wei, Guohui Fu, Ting Sun. <em>Acta Biochimica et Biophysica Sinica</em>, Volume 50, Issue 7, July 2018.