FAM-FLICA® Caspase-1 Assay Kit

$211.00$592.00

Detect caspase-1 activity with the FAM FLICA Caspase-1 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe FAM-YVAD-FMK to label active caspase-1 enzyme in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

Catalog # Size Quantity Price
97 25 Tests $211.00
98 100 Tests $592.00
Catalog #: 97, 98 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-1 probe allows researchers to assess caspase-1 activation.

The FLICA reagent FAM-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the FAM-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-YVAD-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-1 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.

Suspension cells were treated with an apoptosis-inducing agent or DMSO, a negative control, for 4 hours, washed twice, then incubated with ICT’s green caspase-1 inhibitor probe, FAM-YVAD-FMK, for 1 hour and examined under a fluorescence microscope. The top images reveal several experimental cells, all of which fluoresce green, therefore they have active caspase-1. The lower brightfield image at right reveals many control cells in the field of view, however the corresponding fluorescence image is dark (lower left); none of these cells have active caspase-1 (Dr. Brian W. Lee, ICT).caspase1data_1

• Reagent name: FAM-YVAD-FMK
• Target: Caspase-1
• 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 97: 25 Tests
• FLICA Caspase-1 Reagent (FAM-YVAD-FMK), 1 vial, #655
• 10X Apoptosis Wash Buffer, 15 mL, #635
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 98: 100 Tests
• FLICA Caspase-1 Reagent (FAM-YVAD-FMK), 4 vials, #655
• 10X Apoptosis Wash Buffer, 60 mL, #634
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Laencina L, Dubois V, Le Moigne V, Viljoen A, Majlessi L, Pritchard J, Bernut A, Piel L, Roux AL, Gaillard JL, Lombard B, Loew D, Rubin EJ, Brosch R, Kremer L, Herrmann JL, Girard-Misguich F. Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus. Proc Natl Acad Sci U S A. 2018. Jan 30;115(5):E1002-E1011. doi: 10.1073/pnas.1713195115. Epub 2018 Jan 17. Abstract

“The FAM FLICA Caspase-1 Assay Kit (ImmunoChemistry Technologies) was used to evaluate the presence of catalytically active forms of caspase 1 p10 and p12.”

Mantegazza AR, Wynosky-Dolfi MA, Casson CN, Lefkovith AJ, Shin S, Brodsky IE, Marks MS. Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity. PLoS Pathog. 2017. Dec 18;13(12):e1006785. doi: 10.1371/journal.ppat.1006785. eCollection 2017 Dec. Full text

“The caspase-1 probe FAM-YVAD-FMK (FAM-FLICA) was from ImmunoChemistry Technologies (Bloomington, MN). Recombinant LLO was purified from E. coli strain DP-3570 expressing six histidine (His6)-tagged LLO (kindly provided by Daniel Portnoy, University of California, Berkeley), and was coated onto 3-micron polystyrene beads (Polysciences Inc.) as previously described [81, 82].”

Clerc P, Jeanjean P, Hallalli N, Gougeon M, Pipy B, Carrey J, Fourmy D, Gigoux V. Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death. J Control Release. 2017. Dec 1;270:120-134. doi: 10.1016/j.jconrel.2017.11.050. [Epub ahead of print]. Abstract

” … for 2 h. Immediately, 3 or 17 h after AMF exposure, cells were incubated with Fluorochrome-Labeled Inhibitors of Caspase-1 (FLICA, FAM-YVAD-FMK, excitation: 488 nm) or Caspase-3 (FLICA, FAM-DEVD-FMK, excitation: 488 nm) reagent (Immunochemistry …”

Mendonça R, Ferro KP, Leonardo FC, Silva JA, Pericole FV, Saad ST, Costa FF, Conran N. Canonical Inflammasome Formation in Monocytes of Sickle Cell Anemia Patients. Blood. 2017. 130:2233. Abstract

“Caspase-1 activity was determined in phenotypically-characterized monocyte populations by flow cytometry (Fam-FlicaTM kit, Immunochemistry Technologies).”

Bruder-Nascimento T, Ferreira NS, Zanotto CZ, Ramalho F, Pequeno IO, Olivon VC, Neves KB, Alves-Lopes R, Campos E, Silva CA, Fazan R, Carlos D, Mestriner FL, Prado D, Pereira FV, Braga T, Luiz JP, Cau SB, Elias PC, Moreira AC, Câmara NO, Zamboni DS, Alves-Filho JC, Tostes RC. NLRP3 Inflammasome Mediates Aldosterone-Induced Vascular Damage. Circulation. 134(23): 1866-1880. Abstract.

“BMDMs cells were stained for 1 hour with a FAM-FLICA caspase-1 assay (FAM-YVAD-FMK) kit (Immunochemistry Technologies, Bloomington, MN) according to the manufacturers instructions.”

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.”

Bednash JS, Weathington N, Londino J, Rojas M, Gulick DL, Fort R, Han S, McKelvey AC, Chen BB, and Mallampalli RK. Targeting the deubiquitinase STAMBP inhibits NALP7 inflammasome activity. Nat Commun. 2017. May 11;8:15203. doi: 10.1038/ncomms15203. Full text.

“THP-1 cells (1 x 10^6 cells per mL) were pre-treated with BC-1471 (10 mg/mL) for 16 h prior to addition of LPS (500 ng/ml), Pam3CSK4 (100 ng/ml) or control for 6 h. FAM-FLICA reagent (Immunochemistry Technologies, 98) was added 4 h before the end of the assay, prepared according to the manufacturer’s protocol.”

Ferris ST, Zakharov PN, Wan X, Calderon B, Artyomov MN, Unanue ER, and Carrero JA. The islet-resident macrophage is in an inflammatory state and senses microbial products in blood. J. Exp. Med. 2017. Jun 19. pii: jem.20170074. doi: 10.1084/jem.20170074. [Epub ahead of print]. Abstract.

“Islets were isolated and dispersed as described previously. Cells were incubated for 30 min at 37°C with the fluorescent inhibitor probe FAM-YVAD-FMK (Immunochemistry Technologies) to label active caspase-1 enzyme.”

Swan ZD, Bouwer AL, Wonderlich ER, Barratt-Boyes SM. Persistent accumulation of gut macrophages with impaired phagocytic function correlates with SIV disease progression in macaques. Eur. J. Immunol. 2017 Jul 1. doi: 10.1002/eji.201646904. [Epub ahead of print]. Abstract.

” – … Detection of active caspase-1 was carried out using the FAM-FLICA Caspase-1 Assay Kit (ImmunoChemistry Technologies) per the manufacturer’s instructions. To measure responses to stimulation, freshly isolated intestinal and mesenteric LN single cell…”

Dang EV, McDonald JG, Russell DW, Cyster JG. Oxysterol Restraint of Cholesterol Synthesis Prevents AIM2 Inflammasome Activation. Cell. 2017. Nov 16.171(5):1057-1071.e11. doi: 10.1016/j.cell.2017.09.029. Epub 2017 Oct 12. Summary

“BMDMs (1×105 ) were seeded on round glass coverslips in 24-well plates overnight and then stimulated with LPS for 8 hr along with 150X FLICA reagent (ImmunoChemistry Technologies).”

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).”

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