Microscopic Detection of ASC Inflammasomes in Bone Marrow Derived Macrophages Post Stimulation

Materials and Reagents

1. Microscope Slides (Citotest, catalog number: 198105)

2. 12-well plate (Jet, catalog number: TCP011012)

3. 10 cm cell culture dish (Jet, catalog number: TCD010100)

4. 15/50 ml sterile centrifuge tube (Jet, catalog number: CFT011500)

5. Serological Pipet (JETBIOFIL, catalog number: GSP-010-005/GSP-010-010)

6. 25 cm Cell Scraper (BIOFIL, catalog number: CSC011025)

7. Microscope Cover Glass (NEST, catalog number: 801008, Φ15 mm)

8. 5 ml syringe with 26 G needle

9. 20 ml syringe with 18 G needle

10. Mice (C57/BLJ6, Beijing Vital River Laboratory Animal Technology Co., Ltd)

11. RPMI 1640 (Gibco, catalog number: 31800022, 4°C)

12. DMEM/F12 (Gibco, catalog number: 12500062, 4°C)

13. FBS (Hyclone, catalog number: SH30084.03, -20°C/4°C)

14. Nonessential amino acids (Gibco, catalog number: 111140-050, 4°C)

15. Penicillin-streptomycin (Gibco, catalog number: 15140-122, 4°C)

16. PBS (Gibco, catalog number: 21600-069, 4°C)

17. 4% paraformaldehyde (PFA) (Sangon Biotech, catalog number: E672002-0500, 15-25°C (RT))

18. BSA (Sangon Biotech, catalog number: A500023, 4°C)

19. Anti-ASC (AdipoGen, catalog number: AG-25B-0006, -20°C)

20. Saponin (Sigma, catalog number: 47036, 4°C)

21. Fluorescence conjugated secondary antibody (Alexa Fluor^TM 488 goat anti-Rabbit) (Invivogen, catalog number: A11008, -4°C)

22. LPS (Invivogen, catalog number: tlrl-smlps, -20°C)

23. ATP (Sigma, catalog number: FLAAS, -20°C)

24. X-fect Transfection Reagent (X-fect polymer, X-fect buffer) (Clontech, catalog number: 631318, -20°C)

25. 4’,6-diamidine-2-phenylindole dihydrochloride (DAPI) (CST, catalog number: 8961 S, -20°C)

26. Anti-fluorescence attenuation sealant (Solarbio, catalog number: S2100, 4°C)

27. TWEEN^® 20 (Sigma-Aldrich, catalog number: P2287)

28. Cell-neubauer improved (LW Scientific, 0.0025 mm²)

29. BBL^TM Trypticase^TM Soy Broth (BD, catalog number: 211768, RT)

30. NaCl (Sangon Biotech, catalog number: A501218-0001, RT)

31. Tryptone (OXOID, catalog number: LP0042, RT)

32. Yeast extract (OXOID, catalog number: LP0021, RT)

33. Agar (SbaseBio, catalog number: A010-1.1, RT)

34. TSB solid media (see Recipes)

35. TSB liquid media (see Recipes)

36. LB solid media (see Recipes)

37. LB liquid media (see Recipes)

38. 1% BSA (see Recipes)

39. 0.1% saponin (see Recipes)

40. 0.1% PBST (see Recipes)

Equipment

1. Opthalmic scissors (Beijing Bao Yuan Industrial Technology, catalog number: M-Y003)

2. Opthalmic forceps (Beijing Bao Yuan Industrial Technology, catalog number: M-Y005)

3. Confocal Microscope (ZEISS, model: LSM880)

4. Microscope (ZEISS, model: Primo vert iLED)

5. Biological Safety Cabinets Clean Benches (ThermoFisher Scientific, model: 1300)

6. CO₂ Incubator (ThermoFisher Scientific, model: 3111)

7. Portable Pipet-Aid (Drummond, catalog number: 4-000-201)

8. Diaphragm vacuum pump (Tianjin Jinteng Experiment Equipment, model: GM-0.33A)

9. Nanophotometer p-class (IMPLEN, model: NT-80)

10. -80°C freezer (Thermo, model: FDE30086FV)

Software

1. ZEN black_2-3SP1 (ZEISS, https://www.zeiss.com/corporate/us/home.html)

2. ZEN blue 2.6 (ZEISS, https://www.zeiss.com/corporate/us/home.html)

Procedure

1. Culture of Francisella novicida (U112 strain) and Salmonella Typhimurium (SL1344 strain)

   1. Francisella novicida (U112)

      1. Recovery of Francisella novicida: Streak the frozen Francisella novicida stock onto TSB solid media (with 1% L-acetylcysteine) and place in a 37°C incubator for 24 h.

      2. Culture of Francisella novicida: Place a single colony of Francisella novicida into 5 ml TSB liquid media (with 1% L-acetylcysteine) and grow in 37°C shaker with 200 rpm rotation overnight.

      3. Subculture of Francisella novicida: Transfer 3 ml of overnight cultured Francisella novicida into 6 ml fresh TSB liquid media (with 1% L-acetylcysteine) and incubate at 37°C while shaking at 200 rpm for 2 h.

      4. Measurement of the concentration of bacteria: Use 1 ml of subcultured Francisella novicida to measure the value of OD₆₀₀ with the Nanophotometer p-class (IMPLEN, NT80) and calculate the concentration of bacteria according this equation “1 (OD₆₀₀)=1 × 10⁶ CFU/μl”.

   2. Salmonella Typhimurium (SL1344)

      1. Recovery of Salmonella Typhimurium: Streak the frozen Salmonella Typhimurium stock onto LB solid media and place in a 37°C incubator for 24 h.

      2. Culture of Salmonella Typhimurium: Place a single colony of Salmonella Typhimurium into 3 ml LB liquid media and grow in 37°C shaker with 200 rpm rotation overnight.

      3. Subculture of Salmonella Typhimurium: Transfer 0.5 ml of overnight cultured Salmonella Typhimurium into 2.5 ml fresh LB liquid media and incubate at 37°C while shaking at 200 rpm for 2 h.

      4. Measurement of the concentration of bacteria: Use 1 ml of subcultured Salmonella Typhimurium to measure the value of OD₆₀₀ with the Nanophotometer p-class (IMPLEN, NT80) and calculate the concentration of bacteria according to this equation “1 (OD₆₀₀)=1 × 10⁶ CFU/μl”.




2. Preparation of L929 supernatant

   1. Seed 5 × 10⁵ L929 cells in 10 cm Petri dish.

   2. Culture L929 cells in the incubator (37°C, 5% CO₂) for 7 days in RPMI 1640 media supplemented with 10% FBS.

   3. Collect the supernatant with a pipette into a 50 ml centrifuge tube.

   4. Centrifuge at 1,962 × g for 10 min and then transfer the supernatant with a pipette into a new 50 ml centrifuge tube. Freeze at -80°C.




3. Generation of BMDMS

   1. Sacrifice the mice and spray 75% alcohol on their whole body. Remove the skin and fur from the hind legs and cut off the hind legs after the hip joint. Place in PBS.

   2. Remove the muscle tissue from the legs and cut off both ends from the tibia and femur (Video 1).

      
      
      
      Video 1. Generation of BMDMs. (This video was made at the Kunming Institute of Zoology according to guidelines from the Kunming Institute of Zoology on Animal Care and was approved by the Animal Research Ethics Board of the Kunming Institute of Zoology under protocol SMKX-20,6020.)
      
   
   

   3. Flush the bone marrow (BM) cells with L929 supernatant-containing DMEM/F-12 media (1:3) supplemented with 10% FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin using a 5 ml syringe with a 26 G needle. Aspirate the flushed bone marrow several times through a 20 ml syringe with an 18 G needle to obtain a single cell suspension.

   4. Culture BM cells in the incubator (37°C, 5% CO₂) for 5 days. Use 10 ml media for each Petri dish and add 5 ml of fresh L929 supernatant-containing DMEM/F-12 media to the Petri dish on day 3.




4. Preparation of cell slides

   1. Place autoclaved cover slips on a 12-well plate.

   2. Wash three times with PBS, for 3 mins each time.

   3. Scrape the cells from the culture dish with a cell scraper, resuspend in 10 ml DMEM/F-12 media with 10% FBS, and count cells using a cell counting chamber.

   4. Seed 5 × 10⁵ BMDMs into 12-well plates and incubate at 37°C and 5% CO₂ overnight. Make sure the cell confluency the next day is around 60%.




5. Cell stimulation and fixation

   1. Change media with 500 μl of fresh DMEM/F-12 media without L929 supernatant before treatment.

   2. Treat BMDMs with different conditions, as follows, and incubate at 37°C and 5% CO₂.

      1. Stimulate BMDMs with 2 μl of LPS (500 ng/ml, 4 h) and 2 μl of ATP (5 mM, 20 min).

      2. Transfect BMDMs with dsDNA (2 μl of 1.5 μg vector control plasmid) using the X-fect transfection reagent (X-fect polymer 0.45 μl and X-fect buffer 100 μl) for 15 min.

      3. Infect BMDMs with10 μl of Francisella novicida U112 strain (100 MOI, 12 h).

      4. Infect BMDMs with10 μl of Salmonella Typhimurium (3 MOI, 1 h).

   3. Remove medium by suction from each well and wash three times by gently swirling with PBS.

   4. Remove PBS by suction and fix with commercial 4% paraformaldehyde for 15 min at RT.

   5. Remove paraformaldehyde by suction and wash the fixed BMDMs on the cover slip by gently swirling with 500 µl PBS three times.




6. Immunofluorescence staining

   1. Block and permeabilize the cells in 250-300 µl 1% BSA with 0.1% saponin at 15-25°C (RT) for 1 h.

   2. Remove block buffer by suction and gentle swirling of the cells with PBS three times for 3 min each time.

   3. Incubate the cells in 300 µl primary antibody solution (anti-ASC in 1% BSA, 0.1% saponin, 1:150 dilution) at 4°C overnight.

   4. Wash the cells three times with 500 µl 0.1% PBST on a rocking platform, for 3 min each time.

   5. Stain the cells with fluorescence conjugated secondary antibody in PBS (1:300 dilution) at 15-25°C (RT) for 1 h.

   6. Wash the cells three times with 500 µl 0.1% PBST on a rocking platform, for 3 min each time.

   7. Stain the cells with 250-300 µl DAPI (5 μg/ml, prepared in PBS) at 15-25°C (RT) for 5-10 min.

   8. Wash the cells three times with 0.1% PBST on a rocking platform, for 3 min each time.




7. Immobilization of cover slip

   1. Remove the cover slip from the 12-well plate with tweezers and air dry.

   2. Drop anti-fluorescence attenuation sealant into the cell-containing side.

   3. Attach the cell-containing side of the cover slip to microscope slides (25 × 75 mm). Drop sealant onto the edge of the cover slip to bind the cover slip and microscope slide together and prevent sample drying. Avoid air bubbles and air dry for 10 min at 15-25°C (RT).




8. Confocal scanning and image acquisition

   1. Scan cells on the ZEISS-LSM880 confocal microscope (100×, the oil-immersion lens).

      Perform the examination with the ZEISS-LSM880 confocal laser scanning microscope using the following settings: excitation 488 nm and emission 530 nm for ASC speck detection, normal scanning speed, and frame 1024 × 1024. The ASC speck formation was detected and illustrated in Figure 1. Image acquisition was performed with the software ZEN black_2-3SP1.

   2. Analyze the data by using the software ZEN blue 2.6 (Protocol in supplementary material).

Recipes

1. TSB solid media (1 L)

   30 g BBL^TM Trypticase^TM Soy Broth

   15 g Agar

   Autoclave at 121°C for 15 min.

   Add L-acetylcysteine (1% volume ratio) to the autoclaved media when the temperature is around 60°C.

2. TSB liquid media (1 L)

   30 g BBL^TM Trypticase^TM Soy Broth

   Autoclave at 121°C for 15 min.

   Add L-acetylcysteine (1% volume ratio) to the autoclaved media when the temperature is around 60°C.

3. LB solid media (1 L)

   10 g NaCl

   10 g Tryptone

   5 g Yeast extract

   15 g Agar

   Autoclave at 121°C for 15 min

4. LB liquid media (1 L)

   10 g NaCl

   10 g Tryptone

   5 g Yeast extract

   Autoclave at 121°C for 15 min

5. 1% BSA

   1 g BSA

   100 ml PBS

6. 0.1% saponin

   0.1 g saponin

   100 ml PBS

7. 0.1% PBST

   1 ml TWEEN^® 20

   1 L PBS

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31970896, 31701134, and 81701578).

Competing interests

The authors declare no conflicts of interests.

Ethics

The present study was approved by the institutional review board of the Kunming Institute of Zoology, Chinese Academy of Sciences. Animal experiment in this protocol: SMKX-2016020; validity period: January 2017 to January 2022.

References

1. Agrawal, I. and Jha, S. (2020). Comprehensive review of ASC structure and function in immune homeostasis and disease. Mol Biol Rep 47(4): 3077-3096.
2. Beilharz, M., De Nardo, D., Latz, E. and Franklin, B. S. (2016). Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence. Methods Mol Biol 1417: 145-158.
3. Guo, Y., Li, L., Xu, T., Guo, X., Wang, C., Li, Y., Yang, Y., Yang, D., Sun, B., Zhao, X., Shao, G. and Qi, X. (2020). HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection. J Clin Invest 130(12): 6301-6316.
4. Hoss, F., Rolfes, V., Davanso, M. R., Braga, T. T. and Franklin, B. S. (2018). Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking. Methods Mol Biol 1714: 149-165.
5. Rathinam, V. A. and Fitzgerald, K. A. (2016). Inflammasome Complexes: Emerging Mechanisms and Effector Functions. Cell 165(4): 792-800.
6. 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(1): 455-462.
7. Stutz, A., Horvath, G. L., Monks, B. G. and Latz, E. (2013). ASC speck formation as a readout for inflammasome activation. Methods Mol Biol 1040: 91-101.