Cell Meter™ Multiplexing Live, Apoptotic and Necrotic Cell Detection Kit III *Triple Fluorescence Colors*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Platform
Fluorescence microscope
Excitation | 494 nm (apoptosis) / 350 nm (necrosis) / 613 nm (live) |
Emission | 520 nm (apoptosis) / 461 nm (necrosis) / 631 nm (live) |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | FITC filter (apoptosis) / DAPI filter (necrosis) / Cy5 filter (live) |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds
- Add triple fluorescence working solution (100 µL/sample)
- Incubate at room temperature or 37oC for 30 - 60 minutes
- Analyze with a fluorescence microscope at FITC channel (apoptosis), DAPI channel (necrosis) or Texas Red/Cy5 channel (healthy cells)
Important notes
We treated HeLa cells with staurosporine (SS) for 4 hours at 37oC to induce cell apoptosis. See Figure 1 for details.
Thaw all components to room temperature before beginning protocol.
PREPARATION OF WORKING SOLUTION
Add 10 µL of 100X Annexin V-iFluor™ 488 conjugate (Component A), 5 µL of 200X Nuclear Blue™ DCS1 (Component C) and 5 µL of 200X Cellbrite™ Red (Component D) to 1 mL of Assay Buffer (Component B). The triple fluorescence assay solution is stable for at least 1 hour at room temperature. Note: As the optimal staining conditions may vary depending on different cell types, it’s recommended to determine the appropriate concentration of Component A, C and D individually.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Remove cell culture medium and test compounds after treatment.
- Add 100 µL/well (96-well plate) or 50 µL/well (384-well plate) of triple fluorescence assay solution. Incubate at 37oC for 30 to 60 minutes, protected from light.
- Wash cells with HBSS, PBS or buffer of your choice twice.
- Analyze the apoptotic cells with Annexin V-iFluor™ 488 conjugate under fluorescence microscope with a FITC filter. The green staining (Ex/Em = 494/520 nm) on the plasma membrane indicates the Annexin V-iFluor™ 488 conjugate binding to PS on cell surface. Monitor the fluorescence intensity with a DAPI filter (Ex/Em = 350/461 nm) for necrosis, Texas Red or Cy5 filter (Ex/Em = 613/631 nm) for live cells using a fluorescence microscope (See Figure 1 for details).
Images
![Fluorescence images of HeLa cells labeled with Cell Meter™ Multiplexing Live, Apoptotic and Necrotic Detection Kit *Triple Fluorescence* (Cat#22846). HeLa cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were treated with 0-500 nM staurosporine (SS) at 37<sup>o</sup>C for 4 hours (A-C), or fixed in ethanol (D), then incubated with triple fluorescence assay solution for 1 hour. The fluorescence signal was measured using a fluorescence microscope with a Cy5 filter for healthy cells (Red), FITC filter for apoptotic (Green) and DAPI filter for necrotic cells (Blue), respectively.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-multiplexing-live-apoptotic-and-necrotic-cell-detection-kit-iii-triple-fluorescence-colors%2Ffigure-for-cell-meter-multiplexing-live-apoptotic-and-necrotic-cell-detection-kit-iii-triple-fluorescence-colors_jQmDO.jpg&w=3840&q=75)
Citations
Authors: Takada, Ichiro and Hidano, Shinya and Takahashi, Sayuri and Yanaka, Kaori and Ogawa, Hidesato and Tsuchiya, Megumi and Yokoyama, Atsushi and Sato, Shingo and Ochi, Hiroki and Nakagawa, Tohru and others,
Journal: Journal of Biological Chemistry (2022)
Authors: Nanashima, Naoki and Horie, Kayo and Chiba, Mitsuru and Nakano, Manabu and Maeda, Hayato and Nakamura, Toshiya
Journal: Molecular Medicine Reports (2017): 6134--6141
Authors: Riaz, Muhammad Assad and Stammler, Angelika and Borgers, Mareike and Konrad, Lutz
Journal: American Journal of Translational Research (2017): 1266
Authors: Coleman, Jack and Liu, Rui and Wang, Kathy and Kumar, Arun
Journal: Apoptosis Methods in Toxicology (2016): 77--92
References
Authors: Sisto M, D'Amore M, Caprio S, Mitolo V, Scagliusi P, Lisi S.
Journal: Ann N Y Acad Sci (2009): 407
Authors: Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H.
Journal: Toxicol Lett (2009): 156
Authors: Domart MC, Esposti DD, Sebagh M, Olaya N, Harper F, Pierron G, Franc B, Tanabe KK, Debuire B, Azoulay D, Brenner C, Lemoine A.
Journal: J Hepatol (2009): 881
Authors: Heinrich A, Balszuweit F, Thiermann H, Kehe K.
Journal: Toxicol Lett (2009): 260
Authors: Kreeger PK, M and hana R, Alford SK, Haigis KM, Lauffenburger DA.
Journal: Cancer Res (2009): 8191
Authors: Elders RC, Baines SJ, Catchpole B.
Journal: Vet Immunol Immunopathol (2009): 11
Authors: Meusch U, Rossol M, Baerwald C, Hauschildt S, Wagner U.
Journal: Arthritis Rheum (2009): 2612
Authors: Shi H, Guan SH.
Journal: Liver Int (2009): 349
Authors: Ling S, Luo R, Dai A, Guo Z, Guo R, Komesaroff PA.
Journal: Phytomedicine (2009): 56
Authors: Hong JY, Lebofsky M, Farhood A, Jaeschke H.
Journal: Am J Physiol Gastrointest Liver Physiol (2009): G572
Application notes
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Abbreviation of Common Chemical Compounds Related to Peptides
Annexin V