Materials and Reagents

HEK293T cell line (ATCC^® number: CRL-11268 )
Dulbecco´s Modified Eagle´s medium F12 (DMEM-F12) (Biowest, catalog number: L0090 )
10% (v/v) Fetal Bovine Serum (Lonza, catalog number: DE14-801F )
200 mM L-Glutamine (Lonza, catalog number: 17-605E )
Penicillin and streptomycin (Lonza, catalog number: 17-603E )
Liquid nitrogen
1x DPBS (Life Technologies, Gibco^®, catalog number: 14190-094 )
1x Percoll (see Recipes)
Buffer A (see Recipes)
2x CHAPS (see Recipes)
1x CHAPS (see Recipes)

Equipment

Tissue culture flasks with quick-release screw cap 75 ventilation (SARSTEDT AG, catalog number: 83.1813.002 )
Tissue culture plate with lid, hydrophobic, sterile (SARSTEDT AG, catalog number: 83.1839 )
37 °C, 5% CO₂ cell culture incubator
Table top cooling centrifuge with rotor for 15 ml Falcon tubes (Sigma-Aldrich, catalog number: 3K30 )
Table top cooling microcentrifuge with rotor for 2 ml tubes (HERMLE, catalog number: Z216MK )
Water bath at 37 °C
Syringe (1 ml) (Nipro Syringe, catalog number: SY31SCTU EC )
20 G and 25 G needles (Becton, Dickinson and Company, catalog number: 300600 )
Tissue culture class II laminar flow hood (Telstar Bio II)
Inverted microscope with epifluorescence (Nikon Eclipse Ti)
Bürker counting chamber
Ultrafree-CL low-binding Durapore PVDF membrane (5 µm) (EMD Millipore, catalog number: UFC40S25 )

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.

Wash the cells with warm PBS and detached them with 6 ml of cold PBS by pipetting up and down against the bottom of the flask. Transfer the cell suspension into a 15 ml tube (representative scheme of steps from 1 to 8 is shown in Figure 1).
The cells were pelleted by centrifugation at 2,000 x g for 3 min. Discard supernatant and re-suspended the pellet in 600 µl of cold Buffer-A and transfer it into a 1.5 ml tube.
Cells in buffer A were lysed on ice by passing through a 1 ml syringe with a 20 G needle (10 times) and then through a 25 G needle (20 times). After that, the lysate was freeze in liquid nitrogen (keep in liquid nitrogen around 30-40 sec or until the lysate is frozen) and subsequent thawed in a water bath at 37 °C (repeat this freeze/thaw cycle 3 times). The cell lysate was passed again through a 25 G needle (10 times).
The lysates were then centrifuged at 400 x g for 8 min. The supernatant was recovered and the pellet discarded.
Supernatant was transferred into a 15 ml tube, diluted with 2 volumes of CHAPS buffer 2x and then filtered using a 5 μm centrifugal filters at 2,000 x g during 10 min. The centrifugal filter was discarded and the filtrate kept.
The filtrate was diluted with 1 volume of CHAPS buffer 2x and mixed by gentle pipetting up and down (this step is to lysate organelles such as mitochondria). Afterwards, the diluted lysate was centrifuged at 2,300 x g for 8 min and the pellet was recovered, discarding the supernatant.
The pellet was re-suspended in 1 ml of CHAPS buffer 1x and centrifuged at 5,000 x g for 8 min, discarding the supernatants and keeping the pellet. Repeat this wash step twice.
After the last centrifugation step, the pellet was re-suspended in 1 ml CHAPS buffer 1x and loaded carefully on the top of 200 µl of 40% Percoll (prepared previously in a 1.5 ml tube).

Figure 1. Representative scheme of ASC and NLRP3 (p.D303N) particles purification protocol illustrating steps from 1 to 8
Percoll was centrifuged at 16,000 x g for 10 min (turn off the break is not necessary). The interface layer containing the inflammasome particles was collected slowly and gently by pipetting and transferred into a new 1.5 ml tube and then washed with 1 ml of CHAPS buffer 1x by centrifugation using the same conditions (Figure 2).
The supernatant was removed and the pellet was re-suspended in 100 µl of 1x CHAPS buffer (Figure 2).
Finally, fluorescent particles were quantified in a fluorescence microscope using a Bürker chamber using a 1:10 dilution (this dilution depends on the concentration of recovered ASC or NLRP3 particles). After quantification the volume was adjusted until reach a density of 5 x 10⁵ particles per µl.

Figure 2. Representative scheme of Percoll step (steps 8-10)

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.

Experiment	Asc-YFP (10⁶ particles)	NLRP3 (p.D303N)-YFP (10⁶ particles)
1	4.70	1.50
2	3.81	1.54
3	3.03	2.25
4	3.9	2.15
5	4.92	1.15
Average	4.07	1.72

Figure 3. Representative image of ASC and NLRP3 (p.D303N) fluorescent particles after purification protocol

Notes

In step 2 cell pellet can be frozen at -80 °C (first in liquid nitrogen and then at -80 °C) or proceed with the speck purification protocol.
Note that the supernatant recovered in step 4 after centrifugation is not clear.

Recipes

1x Percoll (to prepare 200 µl)
120 µl 1x CHAPS
80 µl Percoll-Plus
Buffer A
320 mM sucrose
20 mM HEPES-KOH (pH 7.5)
10 mM KCl
1.5 mM MgCl₂
1 mM EDTA
1 mM EGTA
2x CHAPS buffer
40 mM HEPES-KOH (pH 7.5)
10 mM MgCl₂
1 mM EGTA
0.2 mM PMSF
0.2 % CHAPS
Note: 1x CHAPS buffer is prepared by diluting the CHAPS buffer 2x in distilled water.

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

Baroja-Mazo, A., Martín-Sánchez, F., Gomez, A. I., Martínez, C. M., Amores-Iniesta, J., Compan, V., Barberà-Cremades, M., Yagüe, J., Ruiz-Ortiz, E. and Antón, J. (2014). The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15:738-48.
Fernandes-Alnemri, T. and Alnemri, E. S. (2008). Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol 442: 251-270.
Franklin, B. S., Bossaller, L., De Nardo, D., Ratter, J. M., Stutz, A., Engels, G., Brenker, C., Nordhoff, M., Mirandola, S. R. and Al-Amoudi, A. (2014). The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol 15(8): 727-737.
Schroder, K. and Tschopp, J. (2010). The inflammasomes. Cell 140(6): 821-832.