Assessment of Inflammasome Formation by Flow Cytometry

David P. Sester1, Alina Zamoshnikova2, Sara J. Thygesen1, Parimala R. Vajjhala1, Simon O. Cridland1, Kate Schroder2, Katryn J. Stacey2

1 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 2 Institute for Molecular Bioscience, The University of Queensland, Brisbane
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.40
DOI:  10.1002/cpim.13
Online Posting Date:  August, 2016
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Abstract

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow‐sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.

Keywords: ASC; AIM2; flow cytometry; inflammasome; NLRP3

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Formation of Native ASC Specks in Monocytes and Macrophages
  • Basic Protocol 2: Assessment of ASC Speck Formation in HEK293T Cells Reconstituted with Fluorescently Tagged Inflammasome Components
  • Alternate Protocol 1: Assessment of Candidate Inflammasome Initiators by Transient Expression in HEK293T Cells with Stable Expression of ASC‐EGFP
  • Support Protocol 1: Generation of Stable HEK293T Cell Lines Expressing ASC‐EGFP
  • Basic Protocol 3: Assessment of Recruitment of Procaspases to ASC Specks in Reconstituted HEK293 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of Formation of Native ASC Specks in Monocytes and Macrophages

  Materials
  • BMMs differentiated from bone marrow in the presence of 104 U/ml colony stimulating factor 1 (CSF1) for 6 to 10 days
  • Complete RPMI‐1640 (see recipe) unsupplemented and supplemented with 104 U/ml CSF1
  • 10 µg/ml LPS (see recipe)
  • Dulbecco's modified PBS (DPBS), without calcium or magnesium (e.g., Thermo Fisher Scientific, cat. no. 14190)
  • Isolated human PBMCs (or monocytes)
  • 24‐well tissue culture plate coated with poly‐HEMA (see Kuroda et al., )
  • 100 µM nigericin (see recipe)
  • 150 mM ATP (see recipe)
  • 100% high‐grade ethanol
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • Flow BLOCK (see recipe)
  • 2.4G2 hybridoma supernatant (or anti‐mouse CD16/32, e.g., BD Pharmingen)
  • Flow PERM (see recipe)
  • Anti‐ASC antibody (rabbit anti‐ASC [N15]‐R; e.g., Santa‐Cruz, cat. no. sc‐22514‐R)
  • Flow WASH (see recipe)
  • Goat anti‐rabbit (H+L) Alexa Fluor 488 (e.g., Thermo Fisher Scientific, cat. no. A11008)
  • 10‐cm square bacteriological plastic plates (e.g., Sterilin) or 10‐cm bacterial petri dishes
  • Tissue culture incubator, humidified and set at 37°C with 5% CO 2
  • Centrifuge and microcentrifuge
  • Cell counter
  • 1.5‐ml microcentrifuge tubes
  • 15‐ml and 50‐ml polypropylene conical centrifuge tubes
  • 100‐µm Nitex nylon mesh (e.g., Genesee Scientific, cat. no. 57‐103) or equivalent
  • 5‐ml polypropylene or polystyrene round‐bottom tubes as appropriate for flow cytometer
  • Flow cytometer with 488‐nm laser and appropriate filter configuration
  • Software for analysis of flow cytometry
NOTE: If investigating a new cell type, the capacity for ASC speck formation should be confirmed by fluorescence microscopy before use of the flow cytometric protocol described here.

Basic Protocol 2: Assessment of ASC Speck Formation in HEK293T Cells Reconstituted with Fluorescently Tagged Inflammasome Components

  Materials
  • HEK293T or HEK293 cells
  • Complete DMEM with and without antibiotics (see recipe)
  • Mammalian expression vectors (prepared in pEF6 plasmid using the EF‐1α promoter [e.g., Invitrogen]) for:
  • mCherry
  • Human NLRP3‐mCherry or other candidate genes fused to mCherry
  • Human ASC‐EGFP (ASC with EGFP fused to its C‐terminal)
  • OptiMEM (e.g., Thermo Fisher Scientific)
  • Lipofectamine 2000 (e.g., Thermo Fisher Scientific)
  • Cell dissociation buffer (see recipe)
  • Cell counter
  • Tissue culture‐treated flat‐bottom 24‐well plate
  • Tissue culture incubator, humidified and set at 37°C with 5% CO 2
  • Centrifuge with plate‐spinning capacity
  • 100‐µm Nitex nylon mesh (e.g., Genesee Scientific, cat. no. 57‐103) or equivalent
  • 5‐ml polypropylene or polystyrene round‐bottom tubes as appropriate for flow cytometer
  • Flow cytometer with 488‐nm and 561‐nm lasers and appropriate filter configurations
  • Software for analysis and graphing of flow cytometry data

Alternate Protocol 1: Assessment of Candidate Inflammasome Initiators by Transient Expression in HEK293T Cells with Stable Expression of ASC‐EGFP

  Additional Materials (also see protocol 2)
  • HEK293T cells stably expressing ASC‐EGFP (see protocol 4Support Protocol)

Support Protocol 1: Generation of Stable HEK293T Cell Lines Expressing ASC‐EGFP

  Additional Materials (also see protocol 2)
  • Blasticidin (or another antibiotic suitable for the expression vector used)
  • Cell freezing medium: 90% (v/v) HI‐FCS/10% (v/v) DMSO
  • Tissue culture‐treated 10‐cm plates
  • Tissue culture‐treated flat‐bottom 6‐well plates
  • Cell freezing containers (e.g., Thermo Fisher Scientific)
  • Liquid nitrogen, for long‐term storage

Basic Protocol 3: Assessment of Recruitment of Procaspases to ASC Specks in Reconstituted HEK293 Cells

  Materials
  • HEK293 or HEK293T cells
  • Complete DMEM with and without antibiotics (see recipe)
  • Plasmids:
  • pcDNA3.1 empty vector
  • pcDNA3.1 expressing human ASC (pcDNA‐ASC)
  • pcDNA3.1 expressing catalytically inactive procaspase 8 (C360S) with a C‐terminal myc tag (pcDNA‐Casp8[C360S]‐Myc)
  • Additive‐free DMEM or OptiMEM (e.g., Thermo Fisher Scientific)
  • Lipofectamine 2000 (e.g., Thermo Fisher Scientific)
  • Dulbecco's modified PBS (DPBS), without calcium or magnesium (e.g., Thermo Fisher Scientific, cat. no. 14190)
  • 100% ethanol
  • Flow BLOCK (see recipe)
  • Flow PERM (see recipe)
  • Rabbit polyclonal anti‐ASC N‐15 antibody (e.g., Santa Cruz, cat. no. sc‐22514‐R)
  • Mouse anti‐myc 9B11 antibody (e.g., Cell Signaling Technology, cat. no. 2276)
  • Flow WASH (see recipe)
  • Goat anti‐mouse (H+L) Alexa Fluor 647 (e.g., Thermo Fisher Scientific, cat. no. A21236)
  • Goat anti‐rabbit (H+L) Alexa Fluor 488 (e.g., Thermo Fisher Scientific, cat. no. A11008)
  • Tissue culture‐treated flat‐bottom 12‐well plates
  • Tissue culture incubator, humidified and set at 37°C with 5% CO 2
  • Centrifuge with plate‐spinning capacity
  • 15‐ml polypropylene conical centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • 100‐µm Nitex nylon mesh (e.g., Genesee Scientific, cat. no. 57‐103) or equivalent
  • 5‐ml polypropylene or polystyrene round‐bottom tubes as appropriate for flow cytometer
  • Flow cytometer with 488‐nm and 640‐nm lasers and appropriate filter configurations
  • Software for analysis and graphing of flow cytometry data
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Figures

Videos

Literature Cited

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  Netea, M.G., Simon, A., van de Veerdonk, F., Kullberg, B.J., Van der Meer, J.W., and Joosten, L.A. 2010. IL‐1beta processing in host defense: Beyond the inflammasomes. PLoS Pathog. 6:e1000661. doi: 10.1371/journal.ppat.1000661.
  Ramdzan, Y.M., Polling, S., Chia, C.P., Ng, I.H., Ormsby, A.R., Croft, N.P., Purcell, A.W., Bogoyevitch, M.A., Ng, D.C., Gleeson, P.A., and Hatters, D.M. 2012. Tracking protein aggregation and mislocalization in cells with flow cytometry. Nat. Methods 9:467‐470. doi: 10.1038/nmeth.1930.
  Sagulenko, V., Thygesen, S.J., Sester, D.P., Idris, A., Cridland, J.A., Vajjhala, P.R., Roberts, T.L., Schroder, K., Vince, J.E., Hill, J.M., Silke, J., and Stacey, K.J. 2013. AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ. 20:1149‐1160. doi: 10.1038/cdd.2013.37.
  Schroder, K. and Tschopp, J. 2010. The inflammasomes. Cell 140:821‐832. doi: 10.1016/j.cell.2010.01.040.
  Sester, D.P., Thygesen, S.J., Sagulenko, V., Vajjhala, P.R., Cridland, J.A., Vitak, N., Chen, K.W., Osborne, G.W., Schroder, K., and Stacey, K.J. 2015. A novel flow cytometric method to assess inflammasome formation. J. Immunol. 194:455‐462. doi: 10.4049/jimmunol.1401110.
  Stacey, K.J., Idris, A., Sagulenko, V., Vitak, N., and Sester, D.P. 2016. Methods for delivering DNA to intracellular receptors. In Toll‐Like Receptors: Practice and Methods (C.E. McCoy, ed.) pp. 93‐106. Springer, New York. doi: 10.1007/978‐1‐4939‐3335‐8.
  Vajjhala, P.R., Lu, A., Brown, D.L., Pang, S.W., Sagulenko, V., Sester, D.P., Cridland, S.O., Hill, J.M., Schroder, K., Stow, J.L., Wu, H., and Stacey, K.J. 2015. The inflammasome adaptor ASC induces procaspase‐8 death effector domain filaments. J. Biol. Chem. 290:29217‐29230. doi: 10.1074/jbc.M115.687731.
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  Yin, Q., Sester, D.P., Tian, Y., Hsiao, Y.S., Lu, A., Cridland, J.A., Sagulenko, V., Thygesen, S.J., Choubey, D., Hornung, V., Walz, T., Stacey, K.J., and Wu, H. 2013. Molecular mechanism for p202‐mediated specific inhibition of AIM2 inflammasome activation. Cell Rep. 4:327‐339. doi: 10.1016/j.celrep.2013.06.024.
Key References
  Sester et al., 2015. See above.
  This paper details the development, validation, and original applications of the TOFIE method.
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