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Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*

FAM-YVAD-FMK fluorometric detection of active caspase 1 using Kit #20108 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-YVAD-FMK for 1 hour at 37°C. The Fluorescence intensity (300, 000 cells/ 100 μL/well) was measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) with a FlexStation microplate reader using bottom read mode.
FAM-YVAD-FMK fluorometric detection of active caspase 1 using Kit #20108 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-YVAD-FMK for 1 hour at 37°C. The Fluorescence intensity (300, 000 cells/ 100 μL/well) was measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) with a FlexStation microplate reader using bottom read mode.
Ordering information
Price ()
Catalog Number20108
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
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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. This Cell Meter™ Live Cell Caspase 1 Activity Assay Kit is designed to detect cell apoptosis by measuring caspase 1 activation in live cells. It is used for the quantification of activated caspase 1 activities in apoptotic cells, or for screening caspase 1 inhibitors. FAM-YVAD-FMK, the green label reagent, allows for direct detection of activated caspase 1 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

ExcitationFL1 channel
EmissionFL1 channel
Instrument specification(s)FL2 channel for propidium iodide staining

Fluorescence microscope

ExcitationFITC channel
EmissionFITC Channel
Recommended plateBlack wall/clear bottom
Instrument specification(s)TRITC channel for propidium iodide staining, DAPI channel for Hoechst staining

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: FAM-YVAD-FMK1 vial
Component B: Washing Buffer1 bottle (100 mL)
Component C: 500X Propidium Iodide1 vial (100 µL)
Component D: 500X Hoechst Stain1 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-YVAD-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. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm), fluorescence microscope with FITC filter, or flow cytometer with FL1 channel

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

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

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Culture cells to a density optimal for apoptosis induction according to your specific induction protocol, but not to exceed 2 x 106 cells/ mL. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition.  Here are a few examples for inducing apoptosis in suspension culture:
    1. Treating Jurkat cells with 2 µg/ml camptothecin for 3 hours.
    2. Treating Jurkat cells with 1 µM staurosporine for 3 hours.
    3. Treating HL-60 cells with 4 µg/ml camptothecin for 4 hours.
    4. Treating 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 150X FAM-YVAD-FMK stock solution 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-YVAD-FMK labeling. 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 FAM-YVAD-FMK. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.

  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-YVAD-FMK is fluorescent, thus it is important to wash out any unbound reagent to eliminate the background. For detached cells, the concentration of cells should be adjusted to 2 - 5 X 105 cells/100 µL aliquot per microtiter plate well.

  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 fluorescence microplate reader at Ex/Em = 490/525 nm (for propidium iodide, Ex/Em = 535/635 nm, for Hoechst dyes, Ex/Em = 350/461 nm).

    For flow cytometry: Monitor the fluorescence intensity using FL1 channel (FL2 channel for propidium iodide staining). Gate on the cells of interest, excluding debris.

    For fluorescence microscope: Place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Observe cells under a fluorescence microscope using FITC channel (FRITC channel for propidium iodide staining, DAPI channel for Hoechst staining).

    For fluorescence microplate reader: Place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Monitor the fluorescence intensity (bottom read mode) with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm). Note: If it is necessary to equilibrate the cell concentrations, adjust the suspension volume for the induced cells to approximate the cell density of the non-induced population. This adjustment step is optional if your cell treatment does not result in a dramatic loss in stimulated cell population numbers.

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

Citations


View all 3 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
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