Abstract
NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) (Schroder and Tschopp, 2010). We and others have reported that upon inflammasome activation functional oligomeric inflammasome particles of NLRP3 and ASC were released from cells, acting as danger signals to amplify inflammation by promoting the activation of caspase-1 extracellularly (Baroja-Mazo et al., 2014; Franklin et al., 2014). Studying the extracellular function of oligomeric ASC and NLRP3 inflammasome particles was possible by purification of recombinant particles of ASC or the constitutively activated NLRP3 mutant associated with cryopyrin-associated periodic syndromes (CAPS, mutation p.D303N), both tagged with the yellow fluorescent protein (YFP) and expressed in HEK293 cells. The purification process was facilitated by the fact that expression of recombinant ASC or mutant NLRP3 in HEK293 cells resulted in their spontaneous aggregation into specks (Baroja-Mazo et al., 2014) and the protocol was originally adapted from Fernandes-Alnemri and Alnemri (2008).
Materials and Reagents
Equipment
Procedure
Perform all the steps on ice and all the centrifugations at 4 °C unless noted otherwise. Start the purification from 107 HEK293 transiently expressing ASC-YFP or stably expressing NLRP3 (p.D303N)-YFP maintained in DMEM: F12 (1:1) supplemented with 10% FCS, 2 mM Glutamax and 1% penicillin-streptomycin.
Representative data
Table 1. Example of expected amounts of fluorescent particles recovered after purification protocol relative to 107 HEK293T cells expressing ASC-YFP or NLRP3 (p.D303N)-YFP. The cells were detached 48 h after transfection (cells expressing ASC-YFP) or until reach 90-100% of confluence per flask (cells stably expressing NLRP3(pD303N). The data shown are from experiments performed in different days.
Figure 3. Representative image of ASC and NLRP3 (p.D303N) fluorescent particles after purification protocol
Notes
Recipes
Acknowledgments
This protocol has been adapted from the previously published paper: Baroja-Mazo et al. (2014). This work was supported by grants from PN I+D+I 2008-2011-Instituto Salud Carlos III-FEDER (PI13/00174) and European Research Council (ERC-2013-CoG 614578). The authors declare no conflict of interests.
References
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