FAM-FLICA® Caspase-1 (YVAD) Assay Kit
- Prepare samples and controls
- Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
- Reconstitute FLICA with 50 μL DMSO.
- Dilute FLICA 1:5 by adding 200 μL PBS.
- Add diluted FLICA to each sample at 1:30 (e.g., add 10 μL to 290 μL of cultured cells).
- Incubate approximately 1 hour.
- Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.
- If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.
- If desired, fix cells.
- 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.
Dufies, O;Doye, A;Courjon, J;Torre, C;Michel, G;Loubatier, C;Jacquel, A;Chaintreuil, P;Majoor, A;Guinamard, RR;Gallerand, A;Saavedra, PHV;Verhoeyen, E;Rey, A;Marchetti, S;Ruimy, R;Czerucka, D;Lamkanfi, M;Py, BF;Munro, P;Visvikis, O;Boyer, L. Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice. Nature Microbiology. 2021 Jan 11; doi: 10.1038/s41564-020-00832-5. Full Text
Böhme J, Martinez N, Li S, Lee A, Marzuki M, Tizazu AM, Ackart D, Frenkel JH, Todd A, Lachmandas E, Lum J, Shihui F, Ng TP, Lee B, Larbi A, Netea MG, Basaraba R, van Crevel R, Newell E, Kornfeld H, Singhal A. Metformin enhances anti-mycobacterial responses by educating CD8+ T-cell immunometabolic circuits. Nat Commun. 2020 Oct 16;11(1):5225. doi: 10.1038/s41467-020-19095-z. Full Text
"The activity of caspase-1 was determined using fluorescent inhibitor of caspase-1 (FAM-Flica Caspase-1 Assay kit, ImmunoChemistry Technologies). Staining procedure was performed according to manufactures protocol. In brief after live/dead and surface antibody staining of lung and spleen single cells, FLICA reagent FAM-YVAD-FMK was provided to single cells and binding to activated caspase-1 was allowed for 1 h at 37 °C in the dark. Stained cells were washed with apoptosis wash buffer, followed by fixation. The green fluorescent was..."
Gaul S, Leszczynska A, Alegre F, Kaufmann B, Johnson CD, Adams LA, Wree A, Damm G, Seehofer D, Calvente CJ, Povero D, Kisseleva T, Eguchi A, McGeough MD, Hoffman HM, Pelegrin P, Laufs U, Feldstein AE. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J Hepatol. 2020 Aug 4;S0168-8278(20)30522-5. doi: 10.1016/j.jhep.2020.07.041. Online ahead of print. Abstract
Chang Y, Zhu J, Wang D, Li H, He Y, Liu K, Wang X, Peng Y, Pan S, Huang K. NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury. J Neuroinflammation. 2020 Jul 23;17(1):219. doi: 10.1186/s12974-020-01879-1. Full Text
Huang Y, Wang H, Hao Y, Lin H, Dong M, Ye J, Song L, Wang Y, Li Q, Shan B, Jiang Y, Li H, Shao Z, Kroemer G, Zhang H, Bai L, Jin T, Wang C, Ma Y, Cai Y, Ding C, Liu S, Pan Y, Jiang W, Zhou R. Myeloid PTEN promotes chemotherapy-induced NLRP3-inflammasome activation and antitumour immunity. Nat Cell Biol. 2020 Jun;22(6):716-727. doi: 10.1038/s41556-020-0510-3. Epub 2020 May 4. Abstract
Hu JJ, Liu X, Xia S, Zhang Z, Zhang Y, Zhao J, Ruan J, Luo X, Lou X, Bai Y, Wang J, Hollingsworth LR, Magupalli VG, Zhao L, Luo HR, Kim J, Lieberman J, Wu H. FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nat Immunol. 2020 May 4. doi: 10.1038/s41590-020-0669-6. Online ahead of print. Abstract
Reis AS, Barboza R, Murillo O, Barateiro A, Peixoto EPM, Lima FA, Gomes VM, Dombrowski JG, Leal VNC, Araujo F, Bandeira CL, Araujo RBD, Neres R, Souza RM, Costa FTM, Pontillo A, Bevilacqua E, Wrenger C, Wunderlich G, Palmisano G, Labriola L, Bortoluci KR, Penha-Gonçalves C, Gonçalves LA, Epiphanio S, Marinho CRF. Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes. Sci Adv. 2020 Mar 4;6(10):eaax6346. doi: 10.1126/sciadv.aax6346. eCollection 2020 Mar. Full Text
Gao J, Peng S, Shan X, Deng G, Shen L, Sun J, Jiang C, Yang X, Chang Z, Sun X, Feng F, Kong L, Gu Y, Guo W, Xu Q, Sun Y. Inhibition of AIM2 inflammasome-mediated pyroptosis by Andrographolide contributes to amelioration of radiation-induced lung inflammation and fibrosis. Cell Death Dis. 2019 Dec 20;10(12):957. doi: 10.1038/s41419-019-2195-8.>Full Text
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
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
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
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
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.
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.
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.
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.
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.
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
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\r
Question: What is the difference between YVAD and WEHD and when is it an advantage using one or the other?
Answer: Our new product offering FAM-WEHD-FMK is similar to our existing FAM-YVAD-FMK Assay. Both of these peptide sequences are known to target caspase 1,4, and 5. The WEHD sequence is thought to be a “better” caspase-1 target, as the kcat/kM rate is higher for WEHD vs YVAD (meaning faster conversion of substrate product by the enzyme). However, please note that if our understanding of how FLICA works is correct, the FLICA probe never actually binds to the enzyme via the YVAD or WEHD sequence, but rather the FMK moiety, then perhaps these faster binding kinetics are something of a moot point. In practice the performance characteristics of the two product are very similar. In our lab they were shown to be virtually indistinguishable. Nevertheless, we decided to carry both options so that customers can select their preferred targeting sequence based on their individual needs and experience.
Question: Customer is not seeing a difference between control and induced cells(induction with LPS+ATP). Can we help with optimization? Parameters: macrophages induced from THP-1 cells, using 50 ng/ml PMA for 48 hr Cells in 12 well plates at 3×10^5 cells/well Three groups: experimental with HIV, Positive Control and Untreated. Given fresh media 24 hrs then added 1 ug/ml LPS for 24 hr then 5 mM ATP for 2 hr
Answer: In our lab, we actually saw a greater response in the THP-1 monocytes (not PMA-primed), we had the greatest response with LPS exposure at 100 ng/mL + 5 mM ATP for 24 hours. In our THP I monocyte studies we found induction levels ranging from 10-30% (average was 26.2%) in 24 hour (LPS/ATP exposure) samples compared to 3-8% in negative controls. When working with THP-1 cells primed with PMA to become macrophage-like, in general we were able to achieve better results with lower LPS concentrations and exposure periods than with the THP-1 monocytes. For instance, exposure to 10ng/mL LPS for 2 hours without any supplemental ATP was sufficient to produce the desired effect. I am a bit concerned that the customer’s use of 1 ug/mL LPS for 24 hours may be too high concentration/exposure period and the susceptible cells are moving through pyroptosis, lysing, and are lost from the positive control sample well prior to even receiving the FAM-YVAD-FMK stain. If this is the case, they are missing the period when more of the positive control cells would be stain positive with FAM-FLICA. I would encourage them to experiment with lower LPS concentrations and exposure periods and see if their results are improved. It is also important to note caspase-1 is rapidly secreted by macrophages after its activation by the inflammasome pathway. Therefore, it turns out macrophages might not be the best cell model for use with this product. We have also been working with nigericin, as an alternate inducing agent.
Question: The component FAM-YVAD-FMK Part#665 vial in the kit is empty. Please help me to solve this problem.
Answer: All of our FLICA products, including FAM-YVAD-FMK, are lyophilized as part of the manufacturing process. The vials contain such a small amount of material (µg quantities) that the green FAM-FLICA reagents are nearly invisible in the amber vials. It may be visible as a slight iridescent sheen on the sides of the vial. Per the instructions in our manual, the FLICA vials are reconstituted in DMSO and diluted into PBS and subsequently diluted into cell culture media for staining cells. In order to check that the FLICA vial contains the proper lyophilized reagent, please check the appearance of the DMSO-reconstituted FLICA reagent. It should be orange in appearance and once diluted 1:5 in PBS, the FAM-FLICA reagent should be yellow in appearance.