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Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

The increase of caspase is measured by Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit (Cat# 22822) in Jurkat Cells treated with Staurosporine.  Jurkat cells were treated with or without 1 µM of Staurosporine for 4 hours before TF5-VAD-FMK and Nuclear Green DCS1 were added. <strong>A</strong>. Binding of TF5-VAD-FMK to Caspase was measured by NovoCyte at Ex/Em=646/659. <strong>B</strong>. The staining of Nuclear Green DCS1 and TF5-VAD-FMK were both measured by NovoCyte and shown in a dot plot. SS: Staurosporine
The increase of caspase is measured by Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit (Cat# 22822) in Jurkat Cells treated with Staurosporine.  Jurkat cells were treated with or without 1 µM of Staurosporine for 4 hours before TF5-VAD-FMK and Nuclear Green DCS1 were added. <strong>A</strong>. Binding of TF5-VAD-FMK to Caspase was measured by NovoCyte at Ex/Em=646/659. <strong>B</strong>. The staining of Nuclear Green DCS1 and TF5-VAD-FMK were both measured by NovoCyte and shown in a dot plot. SS: Staurosporine
Ordering information
Price ()
Catalog Number22822
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 (280 nm)0.027
Extinction coefficient (cm -1 M -1)250000
Excitation (nm)649
Emission (nm)664
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 (280 nm)
0.027
Extinction coefficient (cm -1 M -1)
250000
Excitation (nm)
649
Emission (nm)
664
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 (caspase-1, -3, -4, -5, -6, -7, -8 and -9) activation in living cells. Caspases activation is widely accepted as a reliable indicator for cell apoptosis. Most caspases have substrate selectivity for the peptide sequence Val-Ala-Asp (VAD). This kit uses TF5-VAD-FMK as a fluorogenic indicator for most caspase activity. TF5-VAD-FMK is cell permeable, nontoxic, and irreversibly binds to activated casepase-1, -3, -4, -5, -6, -7, -8 and -9 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

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Components


Component A: 500X TF5-VAD-FMK1 vial (100 µL)
Component B: Assay Buffer1 bottle (50 mL)
Component C: 500X Green-DCS11 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 TF5-VAD-FMK into 0.5 mL of cell solution
  3. Incubate the cells in a 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 660/20 nm filter (APC channel)

Important notes
Thaw all the kit 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 TF5-VAD-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 TF5-VAD-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 (Component B) or growth medium. Note: TF5-VAD-FMK is fluorescent; therefore 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 Green-DCS for dead cells, which can be detected with 530/30 nm filter (FITC channel)

  7. If desired, fix cells.

  8. Monitor the fluorescence intensity using a flow cytometer with 660/20 nm filter (APC channel). Gate on the cells of interest, excluding debris.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (280 nm)0.027
Extinction coefficient (cm -1 M -1)250000
Excitation (nm)649
Emission (nm)664

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (280 nm)
Cell Meter™ Generic Fluorimetric Caspase Activity Assay Kit *Green Fluorescence Optimized for Flow Cytometry*503525750000.90.09

Citations


View all 14 citations: Citation Explorer
Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants
Authors: Wang, Lin and Hu, Xiaofan and Ma, Xiangyu and Ma, Zhensheng and Zhang, Yang and Lu, Yizhao and Li, Xiang and Lei, Wei and Feng, Yafei
Journal: Colloids and Surfaces B: Biointerfaces (2016): 440--452
microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2
Authors: He, Wei and Feng, Jianfang and Zhang, Yan and Wang, Yuanyuan and Zang, Wenqiao and Zhao, Guoqiang
Journal: Laboratory Investigation (2016): 317--324
Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)
Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006
Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)
Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway
Authors: Wang, Lin and Zhao, Xiong and Wei, Bo-yuan and Liu, Yi and Ma, Xiang-yu and Wang, Jian and Cao, Peng-chong and Zhang, Yang and Yan, Ya-bo and Lei, Wei and others, undefined
Journal: Biochimie (2015): 85--93
Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling
Authors: Han, Jianjun and Zhang, Lu and Guo, Hui and Wysham, Weiya Z and Roque, Dario R and Willson, Adam K and Sheng, Xiugui and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Gynecologic oncology (2015): 668--675
IL-7 abrogates the immunosuppressive function of human double-negative T cells by activating Akt/mTOR signaling
Authors: Allgäuer, Andrea and Schreiner, Elisabeth and Ferrazzi, Fulvia and Ekici, Arif B and Gerbitz, Armin and Mackensen, Andreas and Völkl, Simon
Journal: The Journal of Immunology (2015): 3139--3148
LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts
Authors: Zhang, Hui and Sweezey, Neil B and Kaplan, Feige
Journal: American Journal of Physiology-Lung Cellular and Molecular Physiology (2015): L391--L402