FAM-FLICA® Caspase-1 (YVAD) Assay Kit

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.

Xie, Y;Li, X;Deng, W;Nan, N;Zou, H;Gong, L;Chen, M;Yu, J;Chen, P;Cui, D;Zhang, F. Knockdown of USF2 inhibits pyroptosis of podocytes and attenuates kidney injury in lupus nephritis. Journal of molecular histology. 2023 June

Miao, RF;Tu, J. LncRNA CDKN2B-AS1 interacts with LIN28B to exacerbate sepsis-induced acute lung injury by inducing HIF-1α/NLRP3-mediated pyroptosis. The Kaohsiung journal of medical sciences. 2023 June

Li, Z;Shi, Y;Wang, Y;Qi, H;Chen, H;Li, J;Li, L. Cadmium-induced pyroptosis is mediated by PERK/TXNIP/NLRP3 signaling in SH-SY5Y cells. Environmental toxicology. 2023 June

Liu, L;Feng, L;Gao, J;Hu, J;Li, A;Zhu, Y;Zhang, C;Qiu, B;Shen, Z. Parthenolide targets NLRP3 to treat inflammasome-related diseases. International immunopharmacology. 2023 June

Zhou, W;Zhang, H;Huang, L;Sun, C;Yue, Y;Cao, X;Jia, H;Wang, C;Gao, Y. Disulfiram with Cu2+ alleviates dextran sulfate sodium-induced ulcerative colitis in mice. Theranostics. 2023 May

Zhang, Y;Hailati, J;Ma, X;Midilibieke, H;Liu, Z. Ubiquitin-specific protease 11 Aggravates Ischemia-reperfusion-induced Cardiomyocyte Pyroptosis and Injury by Promoting TRAF3 Deubiquitination. Balkan medical journal. 2023 May

Dong, RJ;Li, J;Zhang, Y;Li, JS;Yang, LH;Kuang, YQ;Wang, RR;Li, YY. Thalidomide promotes NLRP3/caspase-1-mediated pyroptosis of macrophages in Talaromyces marneffei infection. Microbial pathogenesis. 2023 May

Karema-Jokinen, V;Koskela, A;Hytti, M;Hongisto, H;Viheriälä, T;Liukkonen, M;Torsti, T;Skottman, H;Kauppinen, A;Nymark, S;Kaarniranta, K. Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients. The Journal of biological chemistry. 2023 May

Johnson, AF;Sands, JS;Trivedi, KM;Russell, R;LaRock, DL;LaRock, CN. Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection. PLoS pathogens. 2023 April

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Brill, A;Bourne, J;Campos, J;Hopkin, S;Whitworth, K;Palis, J;Senis, Y;Rayes, J;Iqbal, A. Megakaryocyte NLRP3 hyperactivation induces anemia and potentiates inflammatory response in mice. Research Square. 2023 March

Hedbrant, A;Engström, C;Andersson, L;Eklund, D;Westberg, H;Persson, A;Särndahl, E. Occupational quartz and particle exposure affect systemic levels of inflammatory markers related to inflammasome activation and cardiovascular disease. Environmental health : a global access science source. 2023 March

Sun, L;Li, X;Luo, Z;Li, M;Liu, H;Zhu, Z;Wang, J;Lu, P;Wang, L;Yang, C;Wang, T;He, H;Li, M;Shu, C;Li, J. Purinergic Receptor P2 × 7 Contributes to Abdominal Aortic Aneurysm Development via Modulating Macrophage Pyroptosis and Inflammation. Translational research : the journal of laboratory and clinical medicine. 2023 March

Cui, Y;Yang, Y;Tao, W;Peng, W;Luo, D;Zhao, N;Li, S;Qian, K;Liu, F. Neutrophil Extracellular Traps Induce Alveolar Macrophage Pyroptosis by Regulating NLRP3 Deubiquitination, Aggravating the Development of Septic Lung Injury. Journal of Inflammation Research. 2023 February

Ramadan, A;Cao, Z;Gadjeva, M;Zaidi, TS;Rathinam, VA;Panjwani, N. The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis. Investigative ophthalmology & visual science. 2023 February

Lee, H;Do, J;Hong, S;Choi, E;Kim, Y. Potential effects of SORBS1 variants on serum immunoglobulin E levels specific to milk intake in relation to prevalence of atopic dermatitis in Korean children. Journal of Allergy and Clinical Immunology. 2023 February

Reyes, M;Leff, SM;Gentili, M;Hacohen, N;Blainey, PC. Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands. Science advances. 2023 February

Manna, S;Maiti, S;Shen, J;Weiss, A;Mulder, E;Du, W;Esser-Kahn, AP. Nanovaccine that activates the NLRP3 inflammasome enhances tumor specific activation of anti-cancer immunity. Biomaterials. 2023 February

Chen, Y;Zhou, C;Bian, Y;Fu, F;Zhu, B;Zhao, X;Zhang, M;Zhou, C;Yao, S;Zhang, Z;Luo, H;Ge, Y;Wu, C;Ruan, H. Cadmium exposure promotes thyroid pyroptosis and endocrine dysfunction by inhibiting Nrf2/Keap1 signaling. Ecotoxicology and environmental safety. 2023 January

Yang, L;Shou, Y;Li, F;Du, J;Zhu, X;Wang, S;Xie, L;Zhou, L;Zhou, J;Yang, Y;Xu, J. The activation of AIM2/Caspase-1/GSDMD pathway contributes to pyroptosis of keratinocytes in Stevens-Johnson syndrome/toxic epidermal necrolysis. Research Square. 2023 January; doi: 10.21203/rs.3.rs-2447588/v1. PDF

Zack, S;Nikolaienko, R;Cook, B;Melki, R;Zima, A;Campbell, E. Vacuole Membrane Protein 1 (VMP1) Restricts NLRP3 Inflammasome Activation by Modulating SERCA Activity and Autophagy. Research Square. 2023 January; doi: 10.21203/rs.3.rs-2508369/v1. PDF

Zhou, ZY;Bai, SJ;Lu, CW;Yang, B;Wang, J;Lian, S;Jian, ZR;Wang, EL;Wang, GX;Liu, T. Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis. Journal of fish diseases. 2023 January; doi: 10.1111/jfd.13746. Article

Hou, L;Zhu, Z;Jiang, F;Zhao, J;Jia, Q;Jiang, Q;Wang, H;Xue, W;Wang, Y;Tian, L. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles alleviated silica induced lung inflammation and fibrosis in mice via circPWWP2A/miR-223-3p/NLRP3 axis. Ecotoxicology and environmental safety. 2023 February; doi: 10.1016/j.ecoenv.2023.114537. Text

Vaher, H;Kingo, K;Kolberg, P;Pook, M;Raam, L;Laanesoo, A;Remm, A;Tenson, T;Alasoo, K;Mrowietz, U;Weidinger, S;Kingo, K;Rebane, A. Skin colonization with S. aureus can lead to increased NLRP1 inflammasome activation in patients with atopic dermatitis. Journal of Investigative Dermatology. 2023 February; doi: 10.1016/j.jid.2023.01.013. Full Text

Meyers, AK;Wang, Z;Han, W;Zhao, Q;Zabalawi, M;Duan, L;Liu, J;Zhang, Q;Manne, RK;Lorenzo, F;Quinn, MA;Song, Q;Fan, D;Lin, HK;Furdui, CM;Locasale, JW;McCall, CE;Zhu, X. Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation. Cell reports. 2023 January; doi: 10.1016/j.celrep.2022.111941. Article

Marques-da-Silva, C;Poudel, B;Baptista, RP;Peissig, K;Hancox, LS;Shiau, JC;Pewe, LL;Shears, MJ;Kanneganti, TD;Sinnis, P;Kyle, DE;Gurung, P;Harty, JT;Kurup, SP. AIM2 sensors mediate immunity to Plasmodium infection in hepatocytes. Proceedings of the National Academy of Sciences of the United States of America. 2023 January; doi: 10.1073/pnas.2210181120. Text

Mitchell, CA;Verovskaya, EV;Calero-Nieto, FJ;Olson, OC;Swann, JW;Wang, X;Hérault, A;Dellorusso, PV;Zhang, SY;Svendsen, AF;Pietras, EM;Bakker, ST;Ho, TT;Göttgens, B;Passegué, E. Stromal niche inflammation mediated by IL-1 signalling is a targetable driver of haematopoietic ageing. Nature cell biology. 2023 January; doi: 10.1038/s41556-022-01053-0. Article

Chao, YY;Puhach, A;Frieser, D;Arunkumar, M;Lehner, L;Seeholzer, T;Garcia-Lopez, A;van der Wal, M;Fibi-Smetana, S;Dietschmann, A;Sommermann, T;Ćiković, T;Taher, L;Gresnigt, MS;Vastert, SJ;van Wijk, F;Panagiotou, G;Krappmann, D;Groß, O;Zielinski, CE. Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation. Nature immunology. 2023 February; doi: 10.1038/s41590-022-01386-w. Article

Liu, W;Yang, J;Fang, S;Jiao, C;Gao, J;Zhang, A;Wu, T;Tan, R;Xu, Q;Guo, W. Spirodalesol analog 8A inhibits NLRP3 inflammasome activation and attenuates inflammatory disease by directly targeting adaptor protein ASC. The Journal of biological chemistry. 2022 November 12; doi: 10.1016/j.jbc.2022.102696 Research Article

Liang, C;Peng, Y;Sun, H;Wang, L;Jiang, L;Zou, S. Silencing lncRNA KCNQ1OT1 reduced hepatic ischemia reperfusion injury-induced pyroptosis by regulating miR-142a-3p/HMGB1 axis. Molecular and Cellular Biochemistry. 2022 October 29; doi: 10.1007/s11010-022-04586-y. Full Article

Jiang, Y;Yang, Y;Hu, Y;Yang, R;Huang, J;Liu, Y;Wu, Y;Li, S;Ma, C;Humphries, F;Wang, B;Wang, X;Hu, Z;Yang, S. Gasdermin D restricts anti-tumor immunity during PD-L1 checkpoint blockade. Cell reports. 2022 October 25; doi: 10.1016/j.celrep.2022.111553. Text

Moraes, C;Hottz, E;Dos Santos Ornellas, D;Adesse, D;de Azevedo, C;d’Avila, J;Zaverucha-do-Valle, C;Maron-Gutierrez, T;Barbosa, H;Bozza, P;Bozza, F. Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1β-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy. Molecular Neurobiology. 2022 October 25; doi: 10.1007/s12035-022-03067-z. Abstract

Hu, R;Liang, J;Ding, L;Zhang, W;Liu, X;Song, B;Xu, Y. Edaravone dexborneol provides neuroprotective benefits by suppressing NLRP3 inflammasome-induced microglial pyroptosis in experimental ischemic stroke. International immunopharmacology. 2022 October 21; doi: 10.1016/j.intimp.2022.109315. Article

Yuan, X;Bhat, OM;Zou, Y;Li, X;Zhang, Y;Li, PL. Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation during Hypercholesterolemia. Journal of lipid research. 2022 October 14; doi: 10.1016/j.jlr.2022.100298. Full Text

Wan, Y;Zhang, W;Huang, C;Jian, J;Zhang, Y;Liu, Q;Chen, P;Zhu, X. Ursolic acid alleviates Kupffer cells pyroptosis in liver fibrosis by the NOX2/NLRP3 inflammasome signaling pathway. International immunopharmacology. 2022 October 14; doi: 10.1016/j.intimp.2022.109321. Text

Wen-Yuan, W;Wan-Qing, Y;Qi-Yun, H;Yu-Si, L;Shao-Jie, Q;Jin-Tao, L;Hui, M;Fang, C;Hui-Ling, Y. mTORC1-Dependent and GSDMD-Mediated Pyroptosis in Developmental Sevoflurane Neurotoxicity. Molecular neurobiology. 2022 October 13; doi: 10.1007/s12035-022-03070-4. Abstract

Sun, Y;Han, R;Wang, J;Qin, Y;Ren, Z;Feng, X;Liu, Q;Wang, X. A single-beam of light priming the immune responses and boosting cancer photoimmunotherapy. Journal of Controlled Release : Official Journal of the Controlled Release Society. 2022 September 8; doi: 10.1016/j.jconrel.2022.08.057. Full Article

He, L;Wei, T;Huang, Y;Zhang, X;Zhu, D;Liu, H;Wang, Z. miR-214-3p Deficiency Enhances Caspase-1-Dependent Pyroptosis of Microglia in White Matter Injury. Journal of Immunology Research. 2022 August 22; doi: 10.1155/2022/1642896. Full Article

Yin, H;Wu, M;Lu, Y;Wu, X;Yu, B;Chen, R;Lu, J;Tong, H. HMGB1-activatied NLRP3 inflammasome induces thrombocytopenia in heatstroke rat. PeerJ. 2022 August 4; doi: 10.7717/peerj.13799. Full Text

Trugilho, MRO;Azevedo-Quintanilha, IG;Gesto, JSM;Moraes, ECS;Mandacaru, SC;Campos, MM;Oliveira, DM;Dias, SSG;Bastos, VA;Santos, MDM;Carvalho, PC;Valente, RH;Hottz, ED;Bozza, FA;Souza, TML;Perales, J;Bozza, PT. Platelet proteome reveals features of cell death, antiviral response and viral replication in covid-19. Cell death discovery. 2022 July 16; doi: 10.1038/s41420-022-01122-1. Full Text

McLemore, AF; Hou, HA; Meyer, BS; Lam, NB; Ward, GA; Aldrich, AL; Rodrigues, MA; Vedder, A; Zhang, L; Padron, E; Vincelette, ND; Sallman, DA; Abdel-Wahab, O; List, AF; McGraw, KL. Somatic gene mutations expose cytoplasmic DNA to co-opt the cGAS-STING-NLRP3 axis in Myelodysplastic syndromes. JCI Insight. 2022 July 5; doi: 10.1172/jci.insight.159430. Article

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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

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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.