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Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

Detection of caspase 3/7 activities using Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit in Jurkat cells. TF2-DEVD-FMK fluorescence intensity was induced with the addition of camptothecin. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with TF2-DEVD-FMK for 1 hour. Response was measured using BD FACSCalibur using FL1 channel.
Detection of caspase 3/7 activities using Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit in Jurkat cells. TF2-DEVD-FMK fluorescence intensity was induced with the addition of camptothecin. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with TF2-DEVD-FMK for 1 hour. Response was measured using BD FACSCalibur using FL1 channel.
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Catalog Number22823
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (280 nm)0.09
Extinction coefficient (cm -1 M -1)75000
Excitation (nm)503
Emission (nm)525
Quantum yield0.9
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Correction Factor (280 nm)
0.09
Extinction coefficient (cm -1 M -1)
75000
Excitation (nm)
503
Emission (nm)
525
Quantum yield
0.9
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 generic caspases 3/7 activation in living cells. Caspase 3/7 activation is widely accepted as a reliable indicator for cell apoptosis. Caspases have substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This kit uses TF2-DEVD-FMK as a fluorogenic indicator for caspase 3/7 activity. TF2-DEVD-FMK is cell permeable, nontoxic, and irreversibly binds to activated casepase 3/7 in apoptotic cells. Once bound to caspases, the red fluorescent reagent is retained within the cell. The binding event prevents the caspases from further catalysis but will not stop apoptosis from proceeding. The reagent will start to react with active caspase enzymes within 15 minutes of addition to the media. The kit provides all the essential components with an optimized assay protocol. It is used for the quantification of most activated caspases activities in apoptotic cells, or for screening caspases inhibitors. The green label allows for direct detection of activated caspases in apoptotic cells by flow cytometry.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Components


Component A: 500X TF2-DEVD-FMK1 vial (100 µL)
Component B: Assay Buffer1 bottle (50 mL)
Component C: 500X Propidium Iodide1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds at a density of 5 × 105 to 1 × 106 cells/mL
  2. Add 1 µL of 500X TF2-DEVD-FMK into 0.5 mL of cell solution
  3. Incubate at 37°C, 5% CO2 incubator for 1 - 4 hours
  4. Pellet the cells and resuspend the cells in 0.5 mL of assay buffer or growth medium
  5. Analyze cells using flow cytometer with 530/30 nm filter (FITC channel)

Important notes
Thaw all the components at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL warm medium or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

  2. Treat cells with test compounds for a desired period of time to induce apoptosis, and create positive and negative controls.

  3. Add 1 µL of 500X TF2-DEVD-FMK (Component A) into the treated cells.

  4. Incubate the cells in a 37°C, 5% CO2 incubator for 1 - 4 hours. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with TF2-DEVD-FMK. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.

  5. Wash and spin the cells twice. Resuspend the cells in 0.5 mL of assay buffer or growth medium. Note: TF2-DEVD-FMK is fluorescent; theforefore it is important to wash out any unbound reagent to remove the background.

  6. If desired, label the cells with a DNA stain (such as propidium iodide or 7-AAD for dead cells).

  7. If desired, fix cells.

  8. Monitor the fluorescence intensity using a flow cytometer with 530/30 nm filter (FITC channel). Gate on the cells of interest, excluding debris.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (280 nm)0.09
Extinction coefficient (cm -1 M -1)75000
Excitation (nm)503
Emission (nm)525
Quantum yield0.9

Citations


View all 29 citations: Citation Explorer
Mahuang Xixin Fuzi decoction ameliorates apoptosis via the mitochondrial-mediated signaling pathway in MCM cells
Authors: Yang, Jia and Sun, Qihui and Qingyun, MA and Yu, Qinhui and Liu, Xiaoyun and Liu, Yanliang and Han, Yuxiu and Yang, Yong and Rong, Rong
Journal: Journal of Ethnopharmacology (2022): 115538
Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7
Authors: Bakar, Siti Aishah Abu and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Azhar, Nur Asna and Mohamad, Noor Muzamil and Ahmad, Nor Hazwani
Journal: BioMed Research International (2022)
The BH3 mimetic ($\pm$) gossypol induces ROS-independent apoptosis and mitochondrial dysfunction in human A375 melanoma cells in vitro
Authors: Haasler, Lisa and Kondadi, Arun Kumar and Tsigaras, Thanos and von Montfort, Claudia and Graf, Peter and Stahl, Wilhelm and Brenneisen, Peter
Journal: Archives of Toxicology (2021): 1349--1365
Disruption of p97/VCP induces autophagosome accumulation, cell cycle arrest and apoptosis in human choriocarcinoma cells
Authors: Desdicioglu, Raziye and Sahin, Cansu and Yavuz, Filiz and Cayli, Sevil
Journal: Molecular Biology Reports (2021): 2163--2171
Prostaglandin E1 Improves Cerebral Microcirculation Through Activation of Endothelial NOS and GRPCH1.
Authors: Liu, Lei and Zhang, Hexi and Shi, Yijun and Pan, Lijian
Journal: Journal of Molecular Neuroscience: MN (2020)
Inhibition of p97/VCP function leads to defective autophagosome maturation, cell cycle arrest and apoptosis in mouse Sertoli cells
Authors: Cayli, Sevil and Sahin, Cansu and Sanci, Tuba Ozdemir and Nakkas, Hilal
Journal: Theriogenology (2020): 196--206
microRNA-144 inhibits cell proliferation and invasion by directly targeting TIGAR in esophageal carcinoma
Authors: Mu, Yushu and Wang, Qifei and Tan, Lei and Lin, Lin and Zhang, Benhua
Journal: Oncology letters (2020): 3079--3088
The potential role of extracellular vesicles in COVID-19 associated endothelial injury and pro-inflammation
Authors: Krishnamachary, Balaji and Cook, Christine and Spikes, Leslie and Chalise, Prabhakar and Dhillon, Navneet K
Journal: medRxiv (2020)
CD5L-induced activation of autophagy is associated with hepatoprotection in ischemic reperfusion injury via the CD36/ATG7 axis
Authors: Li, Junjian and Lin, Wei and Zhuang, Lei
Journal: Experimental and Therapeutic Medicine (2020): 2588--2596