ASC-particle-induced Peritonitis    

Materials and Reagents

1. 5 ml syringes (BD, catalog number: 309646 )
   
2. 25 G x1 ½ needles (BD, catalog number: 305127 )
   
3. Conical tubes (15 ml) (Corning, Falcon^®, catalog number: 352095 )
   
4. C57BL/6 mice, typically of 8-12 weeks old (male or female)
   
5. 1 x 10⁵ pASC-GFP particles generated from stable or transiently expressing HEK293 cells and sorted by flow cytometry as described (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008)
   
6. 1x Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV )
   
7. HEPES
   
8. KOH
   
9. Magnesium chloride (MgCl₂)
   
10. EGTA
    
11. CHAPS
    
12. cOmplete protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001)
    
13. IL-1β ELISA kit (BD, catalog number: 559603 )
    
14. Lysis buffer (see Recipes)
    

Equipment

1. Centrifuge
   
2. Fluorescence microscope
   
3. Flow cytometry
   
4. Surgical instruments such as tweezers and scissors
   
5. Biosafety cabinet

Procedure

1. Isolate pASC-GFP particles from 10⁷ HEK293 cells stably or transiently transfected with ASC-GFP as described previously (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008; Martín-Sánchez et al., 2015) or by flow cytometry.
   
2. Remove media from cells, add DPBS and gently scrape cells. Spin down cells at 1,500 x g for 5 min. Discard supernatant.
   
3. Prepare total cell lysates by hypotonic lysis of cell pellets in 20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1% CHAPS, supplemented with protease inhibitors. Use a syringe with 25 G needle to lyse the cells by syringing several times. Centrifuge cell lysates full speed to obtain cell lysates supernatant.
   
4. Induce aggregation of ASC-GFP by incubation of cell lysates supernatant at 37 °C for 30 min as previously described (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008). The typical number of ASC-GFP particles recovered is between 2 x 10⁵ and 4 x 10⁵ per million cells.
   
5. Add a small amount of supernatant to a microscope slide to confirm ASC polymerization into 1-3 μm aggregates by fluorescence microscopy using a standard GFP excitation/emission filter set (excitation: 484 nm; emission: 507 nm) (Figure 1).
   
6. Sort ASC-GFP particles (1-2 μm) by flow cytometry by gating for small particles based on forward scatter versus side scatter. Next start by gating for FITC positive particles. Later confirm ASC-GFP particles by fluorescent microscopy (Figure 1). Particles can be stored at 4 °C for one year.
   
7. Intraperitoneally inject mice with 1 x 10⁵ pASC-GFP particles or the same volume DPBS for control mice (injection volume approximately 200 μl).
   
8. 4 h later sacrifice mice, cut the skin and expose the peritoneal wall (Video 1).
   
   
   
   Video 1. Peritoneal lavage. The video shows how to wash peritoneal cavity after injection of pASC-GFP particles to measure IL-1β by ELISA.
   
   
9. Inject 4 ml of DPBS into the peritoneal cavity.
   
10. Shake the mouse to wash the peritoneal cavity.
    
11. Use the syringe to recover DPBS injected by gradually pulling out the plunge.
    
12. Remove the needle and add content to a conical tube.
    
13. Spin down the cells at 1,200 x g for 5 min.
    
14. Transfer supernatant to fresh conical tubes and measure IL-1β by ELISA. Typical IL-1β concentrations are up to 2 ng/ml and a representative result is shown in our previous publication (de Almeida et al., 2015).  
    

Representative data

Figure 1. Total cell lysates from stable ASC-GFP-expressing HEK293 cells were GFP-sorted by flow cytometry after inducing ASC polymerization and control (Ctrl) and ASC-GFP (pASC)-containing fractions analyzed by fluorescence microscopy. HEK293 cell lysates were used as a negative control.

Notes

1. ASC-GFP particles should be protected from light until use.

Recipes

1. Lysis buffer
   20 mM HEPES-KOH, pH 7.5
   5 mM MgCl₂
   0.5 mM EGTA
   0.1% CHAPS
   Protease inhibitors
   

Acknowledgments

This protocol was adapted from a previously published study (de Almeida et al., 2015). This work was supported by grants the National Institutes of Health (AI099009, AI120625, AI120618 and AR064349 to C.S., AR066739 to A.D., AI120625 and AI120618 to C.S. and A.D., T32AR007611 to L.d.A. and the American Heart Association 13GRNT17110117 to C.S.).

References

1. Baroja-Mazo, A., Martin-Sanchez, F., Gomez, A. I., Martinez, C. M., Amores-Iniesta, J., Compan, V., Barbera-Cremades, M., Yague, J., Ruiz-Ortiz, E., Anton, J., Bujan, S., Couillin, I., Brough, D., Arostegui, J. I. and Pelegrin, P. (2014). The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15(8): 738-748.
   
2. Bondar, V. S. and Puzyr, A. P. (2000). Use of nanodiamond particles for rapid isolation of recombinant apoobelin from Escherichia coli. Dokl Biochem 373(1-6): 129-131.
   
3. Cai, X., Chen, J., Xu, H., Liu, S., Jiang, Q. X., Halfmann, R. and Chen, Z. J. (2014). Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156(6): 1207-1222.
   
4. de Almeida, L., Khare, S., Misharin, A. V., Patel, R., Ratsimandresy, R. A., Wallin, M. C., Perlman, H., Greaves, D. R., Hoffman, H. M., Dorfleutner, A. and Stehlik, C. (2015). The PYRIN domain-only protein POP1 inhibits inflammasome assembly and ameliorates inflammatory disease. Immunity 43(2): 264-276.
   
5. Fernandes-Alnemri, T., Alnemri, E. S. (2008). Chapter thirteen assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol 442: 251-270.
   
6. Fernandes-Alnemri, T., Wu, J., Yu, J. W., Datta, P., Miller, B., Jankowski, W., Rosenberg, S., Zhang, J. and Alnemri, E. S. (2007). The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9): 1590-1604.
   
7. Franklin, B. S., Bossaller, L., De Nardo, D., Ratter, J. M., Stutz, A., Engels, G., Brenker, C., Nordhoff, M., Mirandola, S. R., Al-Amoudi, A., Mangan, M. S., Zimmer, S., Monks, B. G., Fricke, M., Schmidt, R. E., Espevik, T., Jones, B., Jarnicki, A. G., Hansbro, P. M., Busto, P., Marshak-Rothstein, A., Hornemann, S., Aguzzi, A., Kastenmuller, W. and Latz, E. (2014). The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol 15(8): 727-737.
   
8. Martín-Sánchez, F., Gómez, A. I. and Pelegrín, P. (2015). Isolation of particles of recombinant ASC and NLRP3. Bio-protocol 5(10): e1480.
   
9. Ratsimandresy, R. A., Dorfleutner, A. and Stehlik, C. (2013). An update on PYRIN domain-containing pattern recognition receptors: from immunity to pathology. Front Immunol 4: 440.