Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A comprehensive guide to studying inflammasome activation and cell death

Abstract

Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps enable elegant experimental readouts with which to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone marrow–derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and cell death pathways. Our workflow assesses inflammasome activation via the formation of the apoptosis-associated speck-like protein containing a CARD (ASC) speck; cleavage of caspase-1 and gasdermin D; release of IL-1β, IL-18, caspase-1, and lactate dehydrogenase from the cell; and real-time analysis of cell death by imaging. Analyses take up to ~24 h to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic of protocol workflow.
Fig. 2: Real-time cell death analysis in BMDMs following inflammasome activation.
Fig. 3: ASC speck formation in BMDMs following inflammasome activation.
Fig. 4: Caspase-1 cleavage in BMDMs following inflammasome activation.
Fig. 5: Gasdermin D cleavage in BMDMs following inflammasome activation.
Fig. 6: IL-1β and IL-18 ELISA results from the supernatant of BMDMs following inflammasome activation.
Fig. 7: Lactate dehydrogenase release results from the supernatant of BMDMs following inflammasome activation.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. No datasets were generated or analyzed during the current study. All source data underlying the figures are available from the corresponding author upon request.

References

  1. Kesavardhana, S. & Kanneganti, T. D. Mechanisms governing inflammasome activation, assembly and pyroptosis induction. Int. Immunol. 29, 201–210 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Man, S. M., Karki, R. & Kanneganti, T. D. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 277, 61–75 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Martinon, F., Burns, K. & Tschopp, J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell. 10, 417–426 (2002).

    CAS  PubMed  Google Scholar 

  4. Kanneganti, T. D., Lamkanfi, M. & Nunez, G. Intracellular NOD-like receptors in host defense and disease. Immunity 27, 549–559 (2007).

    CAS  PubMed  Google Scholar 

  5. Liston, A. & Masters, S. L. Homeostasis-altering molecular processes as mechanisms of inflammasome activation. Nat. Rev. Immunol. 17, 208–214 (2017).

    CAS  PubMed  Google Scholar 

  6. Tartey, S. & Kanneganti, T. D. Inflammasomes in the pathophysiology of autoinflammatory syndromes. J. Leukoc. Biol. 107, 379–391 (2020).

    CAS  PubMed  Google Scholar 

  7. Man, S. M. & Kanneganti, T. D. Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat. Rev. Immunol. 16, 7–21 (2016).

    CAS  PubMed  Google Scholar 

  8. Place D. E. & Kanneganti, T. D. Cell death-mediated cytokine release and its therapeutic implications. J. Exp. Med. 216, 1474-1486 (2019).

  9. Brydges, S. D. et al. Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies. J. Clin. Invest. 123, 4695–4705 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hoffman, H. M., Mueller, J. L., Broide, D. H., Wanderer, A. A. & Kolodner, R. D. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat. Genet. 29, 301–305 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Brydges, S. D. et al. Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity. Immunity 30, 875–887 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Sharma, D., Sharma, B. R., Vogel, P. & Kanneganti, T. D. IL-1beta and caspase-1 drive autoinflammatory disease independently of IL-1alpha or caspase-8 in a mouse model of familial mediterranean fever. Am. J. Pathol. 187, 236–244 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chae, J. J. et al. Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1beta activation and severe autoinflammation in mice. Immunity 34, 755–768 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Brenner, M., Ruzicka, T., Plewig, G., Thomas, P. & Herzer, P. Targeted treatment of pyoderma gangrenosum in PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome with the recombinant human interleukin-1 receptor antagonist anakinra. Br. J. Dermatol. 161, 1199–1201 (2009).

    CAS  PubMed  Google Scholar 

  15. Yu, J. W. et al. Pyrin activates the ASC pyroptosome in response to engagement by autoinflammatory PSTPIP1 mutants. Mol. Cell 28, 214–227 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Dierselhuis, M. P., Frenkel, J., Wulffraat, N. M. & Boelens, J. J. Anakinra for flares of pyogenic arthritis in PAPA syndrome. Rheumatol. (Oxf.) 44, 406–408 (2005).

    CAS  Google Scholar 

  17. Karki, R. & Kanneganti, T. D. Diverging inflammasome signals in tumorigenesis and potential targeting. Nat. Rev. Cancer 19, 197–214 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Sharma, D. & Kanneganti, T. D. The cell biology of inflammasomes: mechanisms of inflammasome activation and regulation. J. Cell Biol. 213, 617–629 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Boyden, E. D. & Dietrich, W. F. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat. Genet. 38, 240–244 (2006).

    CAS  PubMed  Google Scholar 

  20. Kanneganti, T. D. et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233–236 (2006).

    CAS  PubMed  Google Scholar 

  21. Sutterwala, F. S. et al. Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24, 317–327 (2006).

    CAS  PubMed  Google Scholar 

  22. Mariathasan, S. et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232 (2006).

    CAS  PubMed  Google Scholar 

  23. Martinon, F., Agostini, L., Meylan, E. & Tschopp, J. Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr. Biol. 14, 1929–1934 (2004).

    CAS  PubMed  Google Scholar 

  24. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006).

    CAS  PubMed  Google Scholar 

  25. Franchi, L. et al. Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages. Nat. Immunol. 7, 576–582 (2006).

    CAS  PubMed  Google Scholar 

  26. Miao, E. A. et al. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat. Immunol. 7, 569–575 (2006).

    CAS  PubMed  Google Scholar 

  27. Miao, E. A. et al. Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc. Natl Acad. Sci. USA 107, 3076–3080 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhao, Y. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011).

    CAS  PubMed  Google Scholar 

  29. Fernandes-Alnemri, T., Yu, J. W., Datta, P., Wu, J. & Alnemri, E. S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Burckstummer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat. Immunol. 10, 266–272 (2009).

    PubMed  Google Scholar 

  32. Roberts, T. L. et al. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science 323, 1057–1060 (2009).

    CAS  PubMed  Google Scholar 

  33. Chae, J. J. et al. The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1beta production. Proc. Natl Acad. Sci. USA 103, 9982–9987 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the pyrin inflammasome. Nature 513, 237–241 (2014).

    CAS  PubMed  Google Scholar 

  35. Karki, R. et al. IRF8 regulates transcription of Naips for NLRC4 inflammasome activation. Cell 173, 920–933 e913 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Nour, A. M. et al. Anthrax lethal toxin triggers the formation of a membrane-associated inflammasome complex in murine macrophages. Infect. Immun. 77, 1262–1271 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jin, T., Curry, J., Smith, P., Jiang, J. & Xiao, T. S. Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase-1. Proteins 81, 1266–1270 (2013).

    CAS  PubMed  Google Scholar 

  38. Zhang, L. et al. Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science 350, 404–409 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Datta, D., McClendon, C. L., Jacobson, M. P. & Wells, J. A. Substrate and inhibitor-induced dimerization and cooperativity in caspase-1 but not caspase-3. J. Biol. Chem. 288, 9971–9981 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Broz, P., von Moltke, J., Jones, J. W., Vance, R. E. & Monack, D. M. Differential requirement for caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing. Cell Host Microbe 8, 471–483 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Ramage, P. et al. Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor. J. Biol. Chem. 270, 9378–9383 (1995).

    CAS  PubMed  Google Scholar 

  42. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).

    CAS  PubMed  Google Scholar 

  43. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

    CAS  PubMed  Google Scholar 

  44. Black, R. A., Kronheim, S. R. & Sleath, P. R. Activation of interleukin-1 beta by a co-induced protease. FEBS Lett. 247, 386–390 (1989).

    CAS  PubMed  Google Scholar 

  45. Kostura, M. J. et al. Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proc. Natl Acad. Sci. USA 86, 5227–5231 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Man, S. M. et al. IRGB10 liberates bacterial ligands for sensing by the AIM2 and caspase-11-NLRP3 inflammasomes. Cell 167, 382–396.e317 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Gurung, P. et al. Toll or interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-beta (TRIF)-mediated caspase-11 protease production integrates toll-like receptor 4 (TLR4) protein- and Nlrp3 inflammasome-mediated host defense against enteropathogens. J. Biol. Chem. 287, 34474–34483 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Balakrishnan, A., Karki, R., Berwin, B., Yamamoto, M. & Kanneganti, T. D. Guanylate binding proteins facilitate caspase-11-dependent pyroptosis in response to type 3 secretion system-negative Pseudomonas aeruginosa. Cell Death Discov. 4, 3 (2018).

    PubMed  Google Scholar 

  49. Malireddi, R. K., Ippagunta, S., Lamkanfi, M. & Kanneganti, T. D. Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes. J. Immunol. 185, 3127–3130 (2010).

    CAS  PubMed  Google Scholar 

  50. Place, D. E. et al. ASK family kinases are required for optimal NLRP3 inflammasome priming. Am. J. Pathol. 188, 1021–1030 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhu, Q., Man, S. M., Karki, R., Malireddi, R. K. S. & Kanneganti, T. D. Detrimental type I interferon signaling dominates protective AIM2 inflammasome responses during Francisella novicida infection. Cell Rep. 22, 3168–3174 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Liu, Z. et al. Role of inflammasomes in host defense against Citrobacter rodentium infection. J. Biol. Chem. 287, 16955–16964 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Kuriakose, T. et al. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci. Immunol. 1, aag2045 (2016).

    PubMed  PubMed Central  Google Scholar 

  54. Zheng, M., Karki, R., Vogel, P. & Kanneganti, T.-D. Caspase-6 is a key regulator of innate immunity, inflammasome activation and host defense. Cell 181, 674–687.e13 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Man, S. M. et al. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection. Nat. Immunol. 16, 467–475 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Briard, B. et al. Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection. Nat. Microbiol. 4, 316–327 (2019).

    CAS  PubMed  Google Scholar 

  57. Karki, R. et al. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17, 357–368 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. van de Veerdonk, F. L. et al. The inflammasome drives protective Th1 and Th17 cellular responses in disseminated candidiasis. Eur. J. Immunol. 41, 2260–2268 (2011).

    PubMed  PubMed Central  Google Scholar 

  59. Malireddi, R. K. S. et al. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. J. Exp. Med. 217, e20191644 (2020).

  60. Gurung, P., Sharma, B. R. & Kanneganti, T. D. Distinct role of IL-1beta in instigating disease in Sharpin(cpdm) mice. Sci. Rep. 6, 36634 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Lukens, J. R. et al. Dietary modulation of the microbiome affects autoinflammatory disease. Nature 516, 246–249 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Gurung, P., Burton, A. & Kanneganti, T. D. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1beta-mediated osteomyelitis. Proc. Natl Acad. Sci. USA 113, 4452–4457 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Ippagunta, S. K. et al. Inflammasome-independent role of apoptosis-associated speck-like protein containing a CARD (ASC) in T cell priming is critical for collagen-induced arthritis. J. Biol. Chem. 285, 12454–12462 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Stienstra, R. et al. Inflammasome is a central player in the induction of obesity and insulin resistance. Proc. Natl Acad. Sci. USA 108, 15324–15329 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Rhoads, J. P. et al. Oxidized low-density lipoprotein immune complex priming of the Nlrp3 Inflammasome Involves TLR and FcgammaR cooperation and is dependent on CARD9. J. Immunol. 198, 2105–2114 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Stancu, I. C. et al. Aggregated Tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded Tau pathology in vivo. Acta Neuropathol. 137, 599–617 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Saito, T. et al. Single App knock-in mouse models of Alzheimer’s disease. Nat. Neurosci. 17, 661–663 (2014).

    CAS  PubMed  Google Scholar 

  68. Zaki, M. H. et al. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 32, 379–391 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Zaki, M. H., Vogel, P., Body-Malapel, M., Lamkanfi, M. & Kanneganti, T. D. IL-18 production downstream of the Nlrp3 inflammasome confers protection against colorectal tumor formation. J. Immunol. 185, 4912–4920 (2010).

    CAS  PubMed  Google Scholar 

  70. Sharma, D. et al. Pyrin inflammasome regulates tight junction integrity to restrict colitis and tumorigenesis. Gastroenterology 154, 948–964 e948 (2018).

    CAS  PubMed  Google Scholar 

  71. Man, S. M. et al. Critical role for the DNA sensor AIM2 in stem cell proliferation and cancer. Cell 162, 45–58 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Amer, A. et al. Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf. J. Biol. Chem. 281, 35217–35223 (2006).

    CAS  PubMed  Google Scholar 

  73. Lamkanfi, M. et al. The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation. J. Immunol. 178, 8022–8027 (2007).

    CAS  PubMed  Google Scholar 

  74. Lamkanfi, M., Malireddi, R. K. & Kanneganti, T. D. Fungal zymosan and mannan activate the cryopyrin inflammasome. J. Biol. Chem. 284, 20574–20581 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Gross, O. Measuring the inflammasome. Methods Mol. Biol. 844, 199–222 (2012).

    PubMed  Google Scholar 

  76. Marim, F. M., Silveira, T. N., Lima, D. S. Jr. & Zamboni, D. S. A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells. PLoS One 5, e15263 (2010).

    PubMed  PubMed Central  Google Scholar 

  77. Tran, T. A. T. et al. Whole blood assay as a model for in vitro evaluation of inflammasome activation and subsequent caspase-mediated interleukin-1 beta release. PLoS One 14, e0214999 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Vrentas, C. E. et al. Inflammasomes in livestock and wildlife: insights into the intersection of pathogens and natural host species. Vet. Immunol. Immunopathol. 201, 49–56 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Pelegrin, P., Barroso-Gutierrez, C. & Surprenant, A. P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage. J. Immunol. 180, 7147–7157 (2008).

    CAS  PubMed  Google Scholar 

  80. Yu, J. W. et al. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization. Cell Death Differ. 13, 236–249 (2006).

    CAS  PubMed  Google Scholar 

  81. Lamkanfi, M. et al. Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes. Mol. Cell. Proteom. 7, 2350–2363 (2008).

    CAS  Google Scholar 

  82. Malireddi, R. K. S. et al. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation. J. Exp. Med. 215, 1023–1034 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Malireddi, R. K. S., Kesavardhana, S. & Kanneganti, T. D. ZBP1 and TAK1: master regulators of NLRP3 inflammasome/pyroptosis, apoptosis, and necroptosis (PAN-optosis). Front. Cell. Infect. Microbiol. 9, 406 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Van Opdenbosch, N. et al. Caspase-1 engagement and TLR-induced c-FLIP expression suppress ASC/caspase-8-dependent apoptosis by inflammasome sensors NLRP1b and NLRC4. Cell Rep. 21, 3427–3444 (2017).

    PubMed  PubMed Central  Google Scholar 

  85. Gurung, P. et al. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes. J. Immunol. 192, 1835–1846 (2014).

    CAS  PubMed  Google Scholar 

  86. Christgen, S., et al Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis). Front. Cell. Infect. Microbiol. 10, 237 (2020).

  87. Samir, P., Malireddi, R.K.S., Kanneganti, T.-D. The PANoptosome: A deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis). Front. Cell. Infect. Microbiol. 10, 238 (2020).

  88. Kesavardhana, S. et al. The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development. J. Biol. Chem. 295, 8325–8330 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Karki, R. et al. Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer. JCI Insight 5, e136720 (2020).

    PubMed Central  Google Scholar 

  90. Thornberry, N. A. et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768–774 (1992).

    CAS  PubMed  Google Scholar 

  91. Cerretti, D. P. et al. Molecular cloning of the interleukin-1 beta converting enzyme. Science 256, 97–100 (1992).

    CAS  PubMed  Google Scholar 

  92. Boucher, D., Chan, A., Ross, C. & Schroder, K. Quantifying caspase-1 activity in murine macrophages. Methods Mol. Biol. 1725, 163–176 (2018).

    CAS  PubMed  Google Scholar 

  93. Boucher, D., Duclos, C. & Denault, J. B. General in vitro caspase assay procedures. Methods Mol. Biol. 1133, 3–39 (2014).

    CAS  PubMed  Google Scholar 

  94. Kaushal, V., Herzog, C., Haun, R. S. & Kaushal, G. P. Caspase protocols in mice. Methods Mol. Biol. 1133, 141–154 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Swacha, P., Gekara, N. O. & Erttmann, S. F. Biochemical and microscopic analysis of inflammasome complex formation. Methods Enzymol. 625, 287–298 (2019).

    PubMed  Google Scholar 

  96. Talley, S. et al. A caspase-1 biosensor to monitor the progression of inflammation in vivo. J. Immunol. 203, 2497–2507 (2019).

    CAS  PubMed  Google Scholar 

  97. Poreba, M., Strózyk, A., Salvesen, G. S. & Drag, M. Caspase substrates and inhibitors. Cold Spring Harb. Perspect. Biol. 5, a008680 (2013).

    PubMed  PubMed Central  Google Scholar 

  98. Sester, D. P. et al. Assessment of inflammasome formation by flow cytometry. Curr. Protoc. Immunol. 114, 14.40.11–14.40.29 (2016).

    Google Scholar 

  99. Mazanek, Z. & Sohn, J. Tracking the polymerization of DNA sensors and inflammasomes using FRET. Methods Enzymol. 625, 87–94 (2019).

    CAS  PubMed  Google Scholar 

  100. Xia, S., Ruan, J. & Wu, H. Monitoring gasdermin pore formation in vitro. Methods Enzymol. 625, 95–107 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Sarhan, J. et al. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection. Proc. Natl Acad. Sci. USA 115, E10888–E10897 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Orning, P. et al. Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 362, 1064–1069 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Broz, P. & Monack, D. M. Measuring inflammasome activation in response to bacterial infection. Methods Mol. Biol. 1040, 65–84 (2013).

    CAS  PubMed  Google Scholar 

  104. Nagata, K. et al. Generation of App knock-in mice reveals deletion mutations protective against Alzheimer’s disease-like pathology. Nat. Commun. 9, 1800 (2018).

    PubMed  PubMed Central  Google Scholar 

  105. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl Acad. Sci. USA 97, 6108–6113 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Nierman, W. C. et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151–1156 (2005).

    CAS  PubMed  Google Scholar 

  107. Kuehne, S. A. et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 (2014).

    CAS  PubMed  Google Scholar 

  108. Brand, D. D., Latham, K. A. & Rosloniec, E. F. Collagen-induced arthritis. Nat. Protoc. 2, 1269–1275 (2007).

    CAS  PubMed  Google Scholar 

  109. Liu, Z. et al. DOCK2 confers immunity and intestinal colonization resistance to Citrobacter rodentium infection. Sci. Rep. 6, 27814 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Franchi, L. et al. Critical role for Ipaf in Pseudomonas aeruginosa–induced caspase-1 activation. Eur. J. Immunol. 37, 3030–3039 (2007).

    CAS  PubMed  Google Scholar 

  111. Werner, J. L. et al. Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus. J. Immunol. 182, 4938–4946 (2009).

    CAS  PubMed  Google Scholar 

  112. Christ, A. et al. Western diet triggers NLRP3-dependent innate immune reprogramming. Cell 172, 162–175.e114 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Cardona, A. E., Huang, D., Sasse, M. E. & Ransohoff, R. M. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat. Protoc. 1, 1947–1951 (2006).

    CAS  PubMed  Google Scholar 

  114. He, Y., Taylor, N. & Bhattacharya, A. Isolation and culture of astrocytes from postnatal and adult mouse brains. Methods Mol. Biol. 1938, 37–47 (2019).

    CAS  PubMed  Google Scholar 

  115. Weischenfeldt, J. & Porse, B. Bone marrow-derived macrophages (BMM): isolation and applications. Cold Spring Harb. Protoc. 2008, pdb.prot5080 (2008).

    Google Scholar 

  116. Roney, K. Bone marrow-derived dendritic cells. Methods Mol. Biol. 1031, 71–76 (2013).

    CAS  PubMed  Google Scholar 

  117. Link, A. J. & LaBaer, J. Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb. Protoc. 2011, 993–994 (2011).

    PubMed  Google Scholar 

  118. Kusumbe, A. P., Ramasamy, S. K., Starsichova, A. & Adams, R. H. Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue. Nat. Protoc. 10, 1904–1914 (2015).

    CAS  PubMed  Google Scholar 

  119. Stutz, A., Horvath, G. L., Monks, B. G. & Latz, E. ASC speck formation as a readout for inflammasome activation. Methods Mol. Biol. 1040, 91–101 (2013).

    CAS  PubMed  Google Scholar 

  120. Simpson, R. J. Homogenization of mammalian tissue. Cold Spring Harb. Protoc. 2010, pdb.prot5455 (2010).

    PubMed  Google Scholar 

  121. Zondag, H. A. [Determination and diagnostic significance of the lactate dehydrogenase isoenzymes]. Jaarb. Kankeronderz. Kankerbestrijd. Ned. [Yearb. Cancer Res. Fight Cancer Neth.] 14, 327–329 (1964).

    CAS  Google Scholar 

  122. Samir, P. et al. DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome. Nature 573, 590–594 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Sharma, D., Malik, A., Guy, C., Vogel, P. & Kanneganti, T. D. TNF/TNFR axis promotes pyrin inflammasome activation and distinctly modulates pyrin inflammasomopathy. J. Clin. Invest. 129, 150–162 (2019).

    PubMed  Google Scholar 

  124. Fink, S. L., Bergsbaken, T. & Cookson, B. T. Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms. Proc. Natl Acad. Sci. USA 105, 4312–4317 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Wickliffe, K. E., Leppla, S. H. & Moayeri, M. Killing of macrophages by anthrax lethal toxin: involvement of the N-end rule pathway. Cell Microbiol. 10, 1352–1362 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Karki, R., Lee, E., Sharma, B. R., Banoth, B. & Kanneganti, T. D. IRF8 regulates Gram-negative bacteria-mediated NLRP3 inflammasome activation and cell death. J. Immunol. 204, 2514–2522 (2020).

    CAS  PubMed  Google Scholar 

  127. Wellington, M., Koselny, K., Sutterwala, F. S. & Krysan, D. J. Candida albicans triggers NLRP3-mediated pyroptosis in macrophages. Eukaryot. Cell 13, 329–340 (2014).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of the Kanneganti lab, both past and present, for their comments and suggestions. Work from our laboratory was supported by the US National Institutes of Health (AI101935, AI124346, AR056296, and CA253095 to T.-D.K.) and the American Lebanese Syrian Associated Charities (to T.-D.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

R.E.T., R.K.S.M., and T.-D.K. conceptualized the manuscript. R.E.T. and R.K.S.M. wrote the first draft. All authors reviewed and approved the final draft of the manuscript.

Corresponding author

Correspondence to Thirumala-Devi Kanneganti.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Andrea Dorfleutner, Florian Schmidt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Zheng, M., Karki, R., Vogel, P. & Kanneganti, T.-D. Cell 181, 674–687.e13 (2020): https://www.cell.com/cell/fulltext/S0092-8674(20)30333-0

Samir, P. et al. Nature 573, 590–594 (2019): https://www.nature.com/articles/s41586-019-1551-2

Man, S. M. et al. Cell 167, 382–396.e17 (2016): https://www.cell.com/cell/fulltext/S0092-8674(16)31245-4

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tweedell, R.E., Malireddi, R.K.S. & Kanneganti, TD. A comprehensive guide to studying inflammasome activation and cell death. Nat Protoc 15, 3284–3333 (2020). https://doi.org/10.1038/s41596-020-0374-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-0374-9

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing