FAM-FLICA® Caspase-9 Assay Kit

$195.00$561.00

Detect caspase-9 activity with the FLICA Caspase-9 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe FAM-LEHD-FMK to label active caspase-9 enzyme in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

Catalog # Size Quantity Price
912 25 Tests $195.00
913 100 Tests $561.00
Catalog #: 912, 913 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 FAM FLICA Caspase-9 assay probe allows researchers to assess caspase-9 activation.

The FLICA reagent FAM-LEHD-FMK enters each cell and irreversibly binds to activated caspase-9. Because the FAM-LEHD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-LEHD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active caspase-9 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 Propidium Iodide or Hoechst 33342 to detect necrosis or changes in nuclear morphology, respectively.

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 (e.g., add 10 μL to 290 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, Propidium Iodide, 7-AAD, or an antibody.
9. If desired, fix cells.
10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM FLICA excites at 492 nm and emits at 520 nm.

If working with adherent cells, please see the manual for additional protocols.

Jurkat cells were treated with 1 µM staurosporine for 3 hours to induce caspase-9 activity (top), or were treated with a control (bottom). Both populations were incubated with ICT’s green FAM-LEHD-FMK FLICA caspase-9 reagent. DIC images were taken of both samples. Almost all cells in the induced sample (top) fluoresce green therefore they have activated caspase-9. None of the control cells (bottom) fluoresce green, therefore they do not have activated caspase-9 (Dr. Brian Lee, ICT).
caspase9data

• Reagent name: FAM-LEHD-FMK
• Target: Caspase-9
• Excitation/Emission: 492 nm / 520 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 912: 25 Tests
• FLICA Caspase-9 Reagent (FAM-LEHD-FMK), 1 vial, #677
• 10X Apoptosis Wash Buffer, 15 mL, #635
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 913: 100 Tests
• FLICA Caspase-9 Reagent (FAM-LEHD-FMK), 4 vials, #677
• 10X Apoptosis Wash Buffer, 60 mL, #634
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Zhai Z, Liu W, Kaur M, Luo Y, Domenico J, Samson JM, Shellman YG, Norris DA, Dinarello CA, Spritz RA, Fujita M. NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma. Oncogene. 2017. Abstract.

“Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN, USA) with a fluorescence plate reader.”

Whang J, Back YW, Lee KI, Fujiwara N, Paik S, Choi CH, Park JK, Kim HJ. Mycobacterium abscessus glycopeptidolipids inhibit macrophage apoptosis and bacterial spreading by targeting mitochondrial cyclophilin D. Cell Death Dis. 2017 Aug 24;8(8):e3012. doi: 10.1038/cddis.2017.420. Article

“For confocal microscopy imaging, DEVD-FMK and LEHD-FMK reagents from the FAM-FLICA caspase detection kit (Immunochemistry Technologies, Bloomington, MN, USA) were used.”

Lin JX, Du N, Li P, Kazemian M, Gebregiorgis T, Spolski R, Leonard WJ. Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nat Commun. 2017. Nov 6;8(1):1320. doi: 10.1038/s41467-017-01477-5. Full Text

“To measure caspase activity in the cells, total splenocytes were incubated at 37 °C for 1 h with fluorescent-labelled caspase inhibitor FAM-VAD-FMK probes (FLICA reagent) for Poly caspases (91), caspase-1 (97), caspases-3, 7 (93), and caspase-9 (912) according to the manufacturer’s instructions (Immunochemistry Technologies, Bloomington, MN, USA). Data were acquired using a FACSCanto II flow cytometer (BD Immunocytometry Systems) and analysed using FlowJo (v9.7.5, Tree Star, Inc., Ashland, OR).”

You may also like…