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. The FLICA 660 Caspase-1 probe allows researchers to assess caspase-1 activation.
The FLICA reagent 660-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the 660-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound 660-YVAD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining far red fluorescent signal is a direct measure of the active caspase-1 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by fluorescence microscopy or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative. Unfixed samples may also be analyzed with Hoechst stain to detect changes in nuclear morphology.
1. Prepare samples and controls.
2. Dilute 10X Cellular 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 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells).
6. Incubate approximately 1 hour.
7. Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells.
8. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
9. If desired, fix cells.
10. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 excites at 660 nm and emits at 680-690 nm.
Jurkat cells were divided into treated and untreated pools and stained with 660-YVAD-FMK. After spiking FLICA into the cell culture media, Jurkat cells were treated with a negative control (left) or Nigericin (20 µM, catalog #6698) to induce caspase-1 activity (right) for 24 hours. Cells were washed 3 times and read on an Accuri C6 flow cytometer. Treatment with the negative control induced caspase-1 activity in only 6.8% of the cell population (left), whereas treatment with Nigericin induced caspase-1 activity in 57.3% of the experimental cells (right). This is a ratio of 8:1.
• Reagent name: 660-YVAD-FMK
• Target: Caspase-1
• Excitation/Emission: 660 nm / 690 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• FLICA Caspase-1 Reagent (660-YVAD-FMK), 1 vial, #6323
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Kit Manual
Shen L, Yang Y, Ou T, Key CC, Tong SH, Sequeira RC, Nelson JM, Nie Y, Wang Z, Boudyguina E, Shewale SV, Zhu X. Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy. J Lipid Res. 2017 Jul 20. pii: jlr.M075879. doi: 10.1194/jlr.M075879. [Epub ahead of print]. Full Text.
“Blood cells were then incubated with FLICA 660-YVAD-FMK (ImmunoChemistry Technologies, #9122) at 1:150 dilution in RPMI-1640 medium plus 5% FBS at 37oC for 1 h, followed by staining for monocytes and neutrophils using a cocktail of antibodies against CD45-APC (BD Pharmingen # 559864)”
Moghaddas F, Llamas R, De Nardo D, Martinez-Banaclocha H, Martinez-Garcia JJ, Mesa-Del-Castillo P, Baker PJ, Gargallo V, Mensa-Vilaro A, Canna S, Wicks IP, Pelegrin P, Arostegui JI, Masters SL. A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever. Ann. Rheum. Dis. 2017. Aug 23. pii: annrheumdis-2017-211473. doi: 10.1136/annrheumdis-2017-211473. [Epub ahead of print]. Abstract
” – … Biotechnology, Dallas, Texas, United States) as previously described. Alternatively, for the detection of active caspase-1, PBMCs were incubated for 20 min with Fluorochrome Inhibitor of Caspases (FLICA)660 reagent (ImmunoChemistry Technologies, Bloomington …”
Swanson KV, Junkins RD, Kurkjian CJ, Holley-Guthrie E, Pendse AA, El Morabiti R, Petrucelli A, Barber GN, Benedict CA, Ting JP. A noncanonical function of cGAMP in inflammasome priming and activation. J. Exp. Med. 2017. Dec 4;214(12):3611-3626. doi: 10.1084/jem.20171749. Epub 2017 Oct 13. Abstract
“When used, active caspase-1 was visualized by the addition of FLICA-660 caspase-1 assay kit (9122; ImmunoChemistry Technologies) for the final hour during the transfection, following the manufacturer’s protocol.”
Mishra SK, Gao YG, Deng Y, Chalfant CE, Hinchcliffe EH, Brown RE. CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation. Autophagy. 2017. Nov 22:1-46. doi: 10.1080/15548627.2017.1393129. [Epub ahead of print]. Abstract
“…Activated CASP1 levels were also assessed by western immuno-blotting of cell supernatant against CASP1-p20-specific antibody as well as with FLICA 660-YVAD-FMK detection reagent (ImmunoChemistry Technology, 9122) which consists of active …”
Köse-Vogel N, Stengel S, Gardey E, Kirchberger-Tolstik T, Reuken PA, Stallmach A, Bruns T. Transcriptional Suppression of the NLRP3 Inflammasome and Cytokine Release in Primary Macrophages by Low-Dose Anthracyclines. Cells. 2019 Dec 28;9(1). pii: E79. doi: 10.3390/cells9010079. Full Text
“In order to measure active caspase-1 and subsequent pyroptosis levels, fluorochrome-labeled inhibitors of caspases (FLICA) 660 Caspase-1 assay kit (Immunochemistry Technologies, Bloomington, MN, USA) was used according to company’s recommendations.”