Product Information
Applications
Biological Information
Assay time1.5 h
Sample TypeIntact cells
Species Reactivity
  • A Broad Range Of Species
Physicochemical Information
Emission max.
Excitation max.
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Storage -20°C
Storage ConditionsUpon arrival store the entire contents of the kit at -20°C.
Protect from Light Protect from light
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsLabeled Caspase-8 Inhibitor (FITC-IETD-FMK), Wash Buffer, Unlabeled Caspase Inhibitor (Z-VAD-FMK), and a user protocol.
Specifications

Documentation

Caspase-8 Detection Kit (FITC-IETD-FMK) SDS

Title

Safety Data Sheet (SDS) 

Caspase-8 Detection Kit (FITC-IETD-FMK) Certificates of Analysis

TitleLot Number
QIA113
User Protocol

Revision02-Febuary-2009 RFH
Form100 Tests
FormatFlow cytometry or fluorescence microscopy
Detection methodFluorescence
Speciesa broad range of species
StorageUpon arrival store the entire contents of the kit at -20°C.
Principles of the assayThis Calbiochem® brand Caspase-8 Detection Kit provides a convenient and sensitive means for detecting activated caspase-8 in living cells. The assay employs a Caspase-8 inhibitor conjugated to FITC, the fluorescent marker FITC-IETD-FMK, is a cell-permeable, non-toxic inhibitor that binds irreversibly to activated caspase-8 and allows the detection of Caspase-8 by the measurement of fluorescence intensity (excitation max.: ~485 nm; emission max.: ~535 nm) in a fluorescent microplate reader, by flow cytometry, or by fluorescence microscopy.
Materials provided• FITC-IETD-FMK (Kit Component No. JA7620-100UL): 1 vial, 100 µl
• Wash Buffer (Kit Component No. JA7621-100ML): 2 bottles, 100 ml
• Z-VAD-FMK (Kit Component No. JA7622-10UL): 1 vial, 10 µl
Detailed protocolCaspase-8 Assay Procedures

Staining

1. Induce apoptosis in cells (1 x 106/ml) as desired. Concurrently incubate a control culture without apoptosis induction. An additional negative control can be prepared by adding the caspase inhibitor Z-VAD-FMK at 1 µl/ml to an induced culture to inhibit caspase activity.
2. Aliquot 300 µl each of the induced and control cultures into eppendorf tubes.
3. Add 1 µl of FITC-IETD-FMK to each tube and incubate for 0.5-1 h in a 37°C incubator with 5% CO2.
4. Centrifuge cells at 3,000 rpm for 5 min and remove the supernatant.
5. Resuspend cells in 0.5 ml of wash buffer and centrifuge again at 3000 rpm for 5 min.
6. Repeat step 5.

Proceed depending on method of analysis.

Quantification by Flow Cytometry

1. For flow cytometric analysis, resuspend cells in 300 µl of wash buffer and place on ice.
2. Analyze samples by flow cytometry using the FL-1 channel.

Detection by Fluorescence Microscopy

1. Resuspend cells in 100 µl of wash buffer.
2. Add one drop of the cell suspension to a microslide and cover with a coverslip.
3. Observe cells under a fluorescence microscope using a FITC filter. Caspase-8 positive cells appear to have brighter green signals, whereas caspase-8 negative control cells show a weaker signal.

Analysis by Fluorescence Plate Reader

1. Resuspend cells in 100 µl of wash buffer.
2. Transfer the cell suspension to each well of the black plate.
3. Measure the fluorescence intensity at Ex. max. 485 nm and Em. max. 535 nm. For control, use wells containing unlabeled cells.
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

Related Products & Applications

Categories

Life Science Research > Cell Analysis > Cell-based Assays > Apoptosis / Cell Death Assays