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SR-FLICA® Caspase-8 Assay Kit


Detect caspase-8 activity with the SR-FLICA Caspase-8 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe SR-LETD-FMK to label active caspase-8 enzymes in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

Catalog # Size Quantity Price
9149 25 Tests $195.00
9150 100 Tests $561.00
Catalog #: 9149, 9150 Categories: , , ,

Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells and tissues.

The FLICA reagent SR-LETD-FMK enters each cell and irreversibly binds to activated caspase-8. Because the SR-LETD-FMK FLICA reagent becomes covalently coupled to the active enzymes, it is retained within the cell, while any unbound SR-LETD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining red fluorescent signal is a direct measure of the active caspase-8 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by a fluorescence plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative included in the kit. Unfixed samples may also be analyzed with Hoechst 33342 to detect changes in nuclear morphology.

1. Prepare samples and controls.
2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
3. Reconstitute FLICA with 50 μL DMSO.
4. Dilute FLICA 1:5 by adding 200 μL PBS.
5. Add diluted FLICA to each sample at 1:30 – 1:60. For example, to stain at 1:30, add 10 μL to 290 μL of cultured cells. To stain at 1:60, add 5 μL to 295 μL of cultured cells.
6. Incubate approximately 1 hour.
7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.
8. If desired, label with additional stains, such as Hoechst, 7-AAD, or an antibody.
9. If desired, fix or embed cells.
10. Analyze with a fluorescence microscope, fluorescence platereader, or flow cytometer. SR-FLICA excites at 550-580 nm and emits at 590-600 nm.

Jurkat cells were grown in suspension to 4 x 105 cells/mL, then divided into two separate TC-flasks. One population, “Non-Induced”, received a DMSO vehicle control (A). The other population, “Induced”, was spiked with 1 μM staurosporine (B). Cells were incubated for 4 hours at 37°C, then stained with ICT’s red SR-FLICA caspase-8 inhibitor, SR-LETD-FMK (kit catalog #9150) for 1 hour at 37°C. After labeling, samples were washed three times and slides were prepared. Fluorescence images were acquired using a Nikon Eclipse 90i microscope equipped with a Hamamatsu Flash 4.0 camera. In the treated sample, cells appear bright red, indicating a high level of caspase-8 activity (B, Induced, right). In the non-induced sample, few red positive cells are visible, indicating minimal caspase-8 activity (A, Non-Induced, left). Data courtesy of Mrs. Tracy Murphy, ICT (220:68, 121815).

• Reagent name: SR-LETD-FMK
• Target: Caspase-8
• Excitation/Emission: 550-580 nm / 590-600 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 9149: 25 Tests
• FLICA Caspase-8 Reagent (SR-LETD-FMK), 1 vial, #6693
• 10X Apoptosis Wash Buffer, 15 mL, #635
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 9150: 100 Tests
• FLICA Caspase-8 Reagent (SR-LETD-FMK), 4 vials, #6693
• 10X Apoptosis Wash Buffer, 60 mL, #634
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Kit Manual

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