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Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*

FAM-DEVD-FMK fluorometric detection of active caspases 3/7 using Kit #20100 in Jurkat cells. The cells were treated with 1 μM staurosporine for 3 hours (Red) while untreated cells were used as a control (Blue). Cells were incubated with FAM-DEVD-FMK for 1 hour at 37°C. The Fluorescent intensity (300, 000 cells/100 μL/well) was measured at Ex/Em = 490/525 nm (cut off at 515 nm) with a FlexStation microplate reader using bottom read mode.
FAM-DEVD-FMK fluorometric detection of active caspases 3/7 using Kit #20100 in Jurkat cells. The cells were treated with 1 μM staurosporine for 3 hours (Red) while untreated cells were used as a control (Blue). Cells were incubated with FAM-DEVD-FMK for 1 hour at 37°C. The Fluorescent intensity (300, 000 cells/100 μL/well) was measured at Ex/Em = 490/525 nm (cut off at 515 nm) with a FlexStation microplate reader using bottom read mode.
Fluorometric detection of active caspases 3/7 using FAM-DEVD-FMK (Cat# 20100) in Jurkat cells. The cells were treated with 1 μM staurosporine for 4 hours (Red) while untreated cells were used as a control (Green). Control and treated cells were incubated with FAM-DEVD-FMK for 1 hour at 37 °C, and then washed once after stain.  Fluorescent intensity was measured with NovoCyte™ 3000 Flow Cytometer FITC channel.
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Price ()
Catalog Number20100
Unit Size
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Additional ordering information
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 (260 nm)0.32
Correction Factor (280 nm)0.178
Extinction coefficient (cm -1 M -1)83000
Excitation (nm)493
Emission (nm)517
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.32
Correction Factor (280 nm)
0.178
Extinction coefficient (cm -1 M -1)
83000
Excitation (nm)
493
Emission (nm)
517
Our Cell Meter™ live cell caspases activity assay kits are based on fluorescent FMK inhibitors of caspases. These inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the caspase inhibitors bind covalently to the active caspases. The activation of caspase 3/7 is important for the initiation of apoptosis. It has been proven that caspase 3/7 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This kit uses FAM-DEVD-FMK as a fluorescent indicator for caspase 3/7 activity. FAM-DEVD-FMK irreversibly binds to activated caspase 3/7 in apoptotic cells. Once bound to caspase 3/7, the fluorescent reagent is retained inside the cell. The binding event inhibits caspase 3/7 but will not stop apoptosis from proceeding. There are a variety of parameters that can be used for monitoring cell apoptosis. This Cell Meter™ Live Cell Caspase 3/7 Activity Assay Kit is designed to detect cell apoptosis by measuring caspase 3/7 activation in live cells. It is used for the quantification of activated caspase 3/7 activities in apoptotic cells, or for screening caspase 3/7 inhibitors. FAM-DEVD-FMK, the green label reagent, allows for direct detection of activated caspase 3/7 in apoptotic cells by fluorescence microscopy, flow cytometer, or fluorescent microplate reader. The kit provides all the essential components with an optimized assay protocol.

Platform


Flow cytometer

Instrument specification(s)See Table 1

Fluorescence microscope

Recommended plateBlack wall/clear bottom
Instrument specification(s)See Table 1

Fluorescence microplate reader

Recommended plateSolid black
Instrument specification(s)See Table 2

Components


Component A: FAM-DEVD-FMK1 vial
Component B: Washing Buffer1 bottle (100 mL)
Component C: 500X Propidium Iodide1 vial (100 µL)
Component D: 500X Hoechst 333421 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds at a density of 5 × 105 to 2 × 106 cells/mL
  2. Add FAM-DEVD-FMK into cell solution at 1:150 ratio
  3. Incubate at room temperature for 1 hour
  4. Pellet the cells, wash and resuspend the cells with buffer or growth medium
  5. Optional: label the cells iwth DNA stain Propidium Iodide or Hoechst 33342 
  6. Analyze the cells at Ex/Em = 490/525 nm

Important notes
Thaw all the components at room temperature before use.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. FAM-DEVD-FMK DMSO stock solution (150X):
Add 50 µL of DMSO to the vial of FAM-DEVD-FMK (Component A).

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Examples for inducing apoptosis in suspension culture:
    Treat Jurkat cells with 2 µg/ml camptothecin for 3 hours
    Treat Jurkat cells with 1 µM staurosporine for 3 hours
    Treat HL-60 cells with 4 µg/ml camptothecin for 4 hours
    Treat HL-60 cells with 1 µM staurosporine for 4 hours. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction

  2. Add 150 X FAM-DEVD-FMK into the cell solution at a 1:150 ratio, and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour. Note: The cells can be concentrated up to ~5 X 106 cells/mL for FAM-DEVD-FMK labeling. The appropriate incubation time depends on the individual cell type and cell concentration used.

  3. Spin down the cells at ~200g for 5 minutes, and wash cells with 1 mL washing buffer (Component B) twice. Resuspend the cells in desired amount of washing buffer. Note: FAM-DEVD-FMK is fluorescent, thus it is important to wash out any unbound reagent to eliminate the background. 

  4. If desired, label the cells with a DNA stain (such as propidium iodide for dead cells, or Hoechst for whole population of the cell nucleus stain).

  5. Monitor the fluorescence intensity by fluorescence microscopy, flow cytometer, or fluorescent microplate reader according to table 1 or table 2. For fluorescence microscopy and fluorescent microplate reader, place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Note: For detached cells, the concentration of cells should be adjusted to 2 - 5 X 105 cells/100 µL aliquot per microtiter plate well. 

Table 1. Fluorescence intensity monitoring for flow cytometry and fluoresence microscopes.

  Flow Cytometry Fluorescence Microscope
FAM-DEVD-FMK FL1 channel FITC channel
Propidium Iodide FL2 channel TRITC channel
Hoechst Dye Violet Laser DAPI channel

Table 2. Fluorescence intensity monitoring for fluorescence microplate readers.

  Excitation Emission Cut Off
FAM-DEVD-FMK 490 nm 525 nm 515 nm
Propidium Iodide 535 nm 635 nm  
Hoechst Dye 350 nm 461 nm  

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.178
Extinction coefficient (cm -1 M -1)83000
Excitation (nm)493
Emission (nm)517

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (280 nm)
Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*5545787500010.179

Citations


View all 4 citations: Citation Explorer
Helicobacter pylori secreted protein HP1286 triggers apoptosis in macrophages via TNF-independent and ERK MAPK-dependent pathways
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: Frontiers in Cellular and Infection Microbiology (2017): 58
Helicobacter pylori Secreted Protein HP1286 Triggers Apoptosis in Macrophages via TNF-Independent and ERK MAPK-Dependent Pathways
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: Frontiers in Cellular and Infection Microbiology (2017): 58
Death receptor 3 mediates necroptotic cell death
Authors: Bittner, Sebastian and Knoll, Gertrud and Ehrenschwender, Martin
Journal: Cellular and Molecular Life Sciences (2016): 1--12
Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: PloS one (2015): e0124407

References


View all 50 references: Citation Explorer
Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation
Authors: Muller I, Lamers MB, Ritchie AJ, Dominguez C, Munoz-Sanjuan I, Kiselyov A.
Journal: Bioorg Med Chem Lett (2011): 5244
Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss
Authors: Abaamrane L, Raffin F, Schmerber S, Sendowski I.
Journal: Eur Arch Otorhinolaryngol (2011): 987
In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk
Authors: Forterre S, Zurbriggen A, Spreng D.
Journal: Vet Immunol Immunopathol (2011): 264
Experimental study on treatment of rabbits optic nerve injury with Caspase-3 inhibitor z-DEVD-fmk
Authors: Zhang W, Yu JG, Wang X, Shen ZS, Zhang JK, Yan H.
Journal: Zhonghua Yan Ke Za Zhi (2010): 1084
Caspase inhibitor ZVAD-fmk facilitates engraftment of donor hematopoietic stem cells in intra-bone marrow-bone marrow transplantation
Authors: Imai Y, Adachi Y, Shi M, Shima C, Yanai S, Okigaki M, Yamashima T, Kaneko K, Ikehara S.
Journal: Stem Cells Dev (2010): 461
Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk
Authors: Lafont E, Milhas D, Teissie J, Therville N, Andrieu-Abadie N, Levade T, Benoist H, Segui B.
Journal: PLoS One (2010): e13638
Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model
Authors: Equils O, Moffatt-Blue C, Ishikawa TO, Simmons CF, Ilievski V, Hirsch E.
Journal: Infect Dis Obstet Gynecol (2009): 749432
Attenuation of allergic contact dermatitis by Z-VAD-FMK, a broad caspase inhibitor: experiment with mice
Authors: Li YY, Yan CL, Xu W.
Journal: Zhonghua Yi Xue Za Zhi (2008): 3153
Effects of Z-FA.FMK on D-galactosamine/tumor necrosis factor-alpha-induced kidney injury and oxidative stress in mice : effects of Z-FA.FMK on TNF-alpha-mediated kidney injury
Authors: Gezginci-Oktayoglu S, Tunali S, Yanardag R, Bolkent S.
Journal: Mol Cell Biochem (2008): 9
Effect of Boc-D-Fmk on hepatocyte apoptosis after bile duct ligation in rat and survival rate after endotoxin challenge
Authors: Sheen-Chen SM, Hung KS, Eng HL.
Journal: J Gastroenterol Hepatol (2008): 1276