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Dec 2020
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Microscopic Detection of ASC Inflammasomes in Bone Marrow Derived Macrophages Post Stimulation
刺激后骨髓源性巨噬细胞中ASC炎症小体的显微检测    

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Abstract

An inflammasome is an intracellular multiprotein complex that plays important roles in host defense and inflammatory responses. Inflammasomes are typically composed of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), cytoplasmic sensor protein, and the effector protein pro-caspase-1. ASC assembly into a protein complex termed ASC speck is a readout for inflammasome activation. Here, we provide a step-by-step protocol for the detection of ASC speck by confocal microscopy in Bone marrow derived macrophages (BMBDs) triggered by chemical stimuli and bacterial pathogens. We also describe the detailed procedure for the generation of BMDMs, stimulating conditions for inflammasome activation, immunofluorescence cell staining of ASC protein, and microscopic examination. Thus far, this method is a simple and reliable manner to visualize and quantify the intracellular localization of ASC speck.


Graphic abstract:



Figure 1. Confocal microscopy detection of ASC speck formation in untreated WT BMDMs and WT BMDMs stimulated with LPS and ATP, transfected with dsDNA, and infected with F. novicida or Salmonella as indicated. Arrow indicates the ASC speck. Scale bars: 10 μm.


Keywords: ASC (ASC), Inflammasome (炎症小体), Fluorescence staining (荧光染色), Confocal microscopy (共聚焦显微镜), NLRP3 (NLRP3), AIM2 (AIM2), NLRC4 (NLRC4)

Background

The innate immune system has a key role in initiating and orchestrating host defense by detecting invading pathogens through membrane-bound and cytosolic pattern recognition receptors (PRRs), which recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Inflammasome activation is an essential innate immune event in response to pathogenic infection and sterile stimuli that causes the initiation of pyroptotic cell death and the release of the proinflammatory cytokines IL-1β and IL-18. Inflammasome assembly is triggered by the activation of upstream sensors, such as NLRP1, NLRP3, AIM2, NLRC4, and PYRIN. Upon activation, sensor proteins form a complex in an ASC-dependent and -independent manner to mediate caspase-1 cleavage and activation. Cleaved caspase-1 in turn leads to the maturation of proinflammatory cytokines IL-1β and IL-18 and the process of GSDMD-mediated pyroptosis (Rathinam and Fitzgerald, 2016). The adaptor ASC protein is composed of a PYRIN domain (PYD) and caspase recruitment domain (CARD), which help ASC function as an adaptor to interact with upstream sensor and effector caspase-1 (Agrawal and Jha, 2020). ASC speck formation is a hallmark of inflammasome activation. Confocal microscopy and flow cytometry are two major methods to detect ASC speck formation (Stutz et al., 2013; Sester et al., 2015; Beilharz et al., 2016; Hoss et al., 2018).


We performed and published the determination of ASC speck formation in BMDMs after the activation of NLRP3, AIM2, and NLRC4 inflammasomes (Guo et al., 2020). In comparison with the method using ASC-GFP fusion protein and flow cytometry, this procedure is able to detect endogenous ASC speck formation and can visualize and quantify subcellular localization of the inflammasome complex with the help of cellular organelle staining. This protocol can be utilized to evaluate any ASC speck formation in other cells after ASC-dependent inflammasome activation.

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 FluorTM 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. BBLTM TrypticaseTM 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 BBLTM TrypticaseTM 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 BBLTM TrypticaseTM 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.

简介

[摘要]一种我nflammasome是一种细胞内的多蛋白复合物对机体的重要作用防御和炎症反应。炎性体通常由包含 CARD (ASC)、细胞质传感器蛋白和效应蛋白 pro-caspase-1 的适配器蛋白凋亡相关斑点样蛋白组成。ASC assembl ÿ成蛋白质复合物地称为ASC斑点为炎性激活的读出。在这里,我们提供了一个步骤一步协议的检测ASC斑点的通过共焦显微镜ý在由化学刺激和细菌病原体触发骨髓衍生的巨噬细胞(BMBDs) 。w ^ ê 还描述了BMDMs 生成的详细过程、炎症体激活的刺激条件、ASC 蛋白的免疫荧光细胞染色和显微镜检查。因此到目前为止,此方法是一种简单且可靠的方式来可视化和量化ASC斑点的细胞内定位。

图文摘要:

图 1.共聚焦显微镜检测未处理的 WT BMDM 和用 LPS 和 ATP 刺激、用 dsDNA 转染并用F. novicida或沙门氏菌感染的WT BMDM 中的 ASC 斑点形成,如图所示。箭头表示 ASC 斑点。比例尺:10 μ m。


[背景]先天免疫系统通过膜结合和细胞溶质模式识别受体 (PRR) 检测入侵病原体,从而在启动和协调宿主防御方面发挥关键作用,PRR 可识别病原体相关和损伤相关的分子模式(PAMP 和 DAMP)。炎症小体激活是响应病原体感染和无菌刺激的重要先天免疫事件,导致细胞焦亡和促炎细胞因子 IL-1 β和 IL-18的释放。炎性组件由上游传感器,诸如NLRP1,NLRP3,AIM2,NLRC4的活化而触发,和PYRIN。在激活时,传感器蛋白形成一个在ASC依赖性和非依赖性方式介导胱天蛋白酶-1切割和激活复合物。裂解的 caspase-1 反过来导致促炎细胞因子 IL-1 β和 IL-18的成熟以及 GSDMD 介导的细胞焦亡过程(Rathinam 和 Fitzgerald,2016)。该适配器ASC蛋白由一个PYRIN结构域(PYD)和胱天蛋白酶募集结构域(CARD),其帮助ASC函数作为适配器与上游传感器和效应胱天蛋白酶-1相互作用(阿格拉瓦尔和贾2020) 。ASC 斑点形成是炎症小体激活的标志。共聚焦显微镜和流式细胞仪是两种主要的方法来检测ASC斑点形成(STUTZ等人,2013; Sester等人。,2015; Beilharz等人。,2016;胡斯等人。,2018 )。
我们执行并发表了在 NLRP3、AIM2 和 NLRC4 炎性体激活后 BMDM 中 ASC 斑点形成的测定(Guo等,2020)。与使用 ASC-GFP 融合蛋白和流式细胞术的方法相比,该程序能够检测内源性 ASC 斑点形成,并可以在细胞器染色的帮助下可视化和量化炎性体复合物的亚细胞定位。该协议可用于评估 ASC依赖性炎症小体激活后其他细胞中的任何 ASC 斑点形成。

关键字:ASC, 炎症小体, 荧光染色, 共聚焦显微镜, NLRP3, AIM2, NLRC4


材料和试剂

 
显微镜载玻片(Citotest,目录号:198105)
12孔板(Jet,目录号:TCP011012)
10 cm细胞培养皿(Jet,目录号:TCD010100)
15/50 ml无菌离心管(Jet,目录号:CFT011500)
血清移液管(JETBIOFIL,目录号:GSP-010-005/GSP-010-010)             
25 cm细胞刮刀(BIOFIL,目录号:CSC011025)             
显微镜盖玻片(NEST,目录号:801008 ,Φ 15毫米)
5 ml 注射器,带 26 G 针头
2 0 ml 注射器,带18 G 针头
小鼠(C57/BLJ6,北京维塔河实验动物科技有限公司)
RPMI 1640(Gibco,目录号:31800022,4°C)
DMEM/F12(Gibco,目录号:12500062,4°C)
FBS(Hyclone,目录号:SH30084.03,-20°C/4°C)
非必需氨基酸(Gibco,目录号:111140-050,4 °C)
青霉素-链霉素(Gibco,目录号:15140-122,4°C)
PBS(Gibco,目录号:21600-069,4°C)
4% 多聚甲醛(PFA)(Sangon Biotech,目录号:E672002-0500,15-25 °C(RT))             
牛血清白蛋白(Sangon Biotech,目录号:A500023,4 °C)
抗 ASC(AdipoGen,目录号:AG-25B-0006,-20°C)
皂苷(Sigma,目录号:47036,4°C)
荧光偶联二抗(Alexa Fluor TM 488 山羊抗兔)(Invivogen,目录号:A11008,-4°C)
LPS(Invivogen,目录号:tlrl-smlps ,-20°C)
ATP(Sigma,目录号:FLAAS,-20°C)
X-fect 转染试剂(X-fect 聚合物,X-fect 缓冲液)(Clontech,目录号:631318,-20°C)
4',6-二脒-2-苯基吲哚二盐酸盐(DAPI)(CST,目录号:8961 S,-20°C)
抗荧光衰减密封剂(Solarbio,目录号:S2100,4 °C)
TWEEN ® 20(Sigma-Aldrich,目录号:P2287)
Cell-neubauer 改进 (LW Scientific, 0.0025 mm 2 )
BBL TM Trypticase TM Soy Broth(BD,目录号:211768,RT)
NaCl(Sangon Biotech,目录号:A501218-0001,RT)
胰蛋白胨(OXOID,目录号:LP0042,RT)
酵母提取物(OXOID,目录号:LP0021,RT)
琼脂(SbaseBio,目录号:A010-1.1,RT)
TSB 固体培养基(见配方)
TSB 液体培养基(见配方)
LB 固体培养基(见配方)
LB 液体培养基(见配方)
1% BSA(见食谱)
0.1% 皂苷(见食谱)
0.1% PBST(见配方)
 
设备
 
眼科剪刀(北京宝源工业科技,目录号:M-Y003)
眼科钳(北京宝源工业科技,目录号:M-Y005)
共聚焦 显微镜(蔡司,型号:LSM880)
显微镜(蔡司,型号:Primo vert iLED)
生物安全柜超净工作台(热˚F isher小号系统求解,型号:1300)
CO 2孵化器(热˚F isher小号系统求解,型号:3111 )
便携式移液器辅助装置(Drummond ,目录号:4-000-201)
隔膜真空泵(天津金腾实验设备,型号:GM-0.33A )
纳米光度计 p 级(IMPLEN,型号:NT-80)
-80°C 冷冻机(The r mo,型号:FDE30086FV )
 
软件
 
ZEN black_2-3SP1(蔡司,https: //www.zeiss.com/corporate/us/home.html )
ZEN blue 2.6(蔡司,https ://www.zeiss.com/corporate/us/home.html )
 
程序
 
的培养弗朗西斯新凶手弗朗西斯菌(U112株)和鼠伤寒沙门氏菌(SL1344 菌株)
新生弗朗西斯菌(U112)
新生弗朗西斯菌的回收:将冷冻的新生弗朗西斯菌原液在 TSB 固体培养基(含 1% L-乙酰半胱氨酸)上划线,并置于 37°C 培养箱中 24 小时。
的培养弗朗西斯新凶手弗朗西斯菌:放置一个的单一菌落弗朗西斯新凶手弗朗西斯菌到5毫升的TSB液体培养基(含1%L-乙酰半胱氨酸)和在37℃下摇床以200rpm旋转生长过夜。
新生弗朗西斯菌的继代培养:将3 ml 过夜培养的新生弗朗西斯菌转移到 6 ml 新鲜 TSB 液体培养基(含 1% L-乙酰半胱氨酸)中,并在 37°C 下孵育,同时以200 rpm摇动2 小时。
细菌的浓度的测定:使用1毫升继代培养的弗朗西斯新凶手弗朗西斯菌到测量电Ë OD的值600与根据此方程“Nanophotometer p级(IMPLEN,NT80)和计算的细菌的浓度为1(OD 600 )= 1 × 10 6 CFU / μ升”。
鼠伤寒沙门氏菌 (SL1344)
鼠伤寒沙门氏菌的回收:将冷冻的鼠伤寒沙门氏菌原液在 LB 固体培养基上划线,然后放入 37°C 培养箱中 24 小时。
的培养沙门氏菌鼠伤寒沙门氏菌:放置一个的单一菌落沙门氏菌鼠伤寒沙门氏菌到3ml LB液体培养基,并生长在37℃下摇床以200rpm旋转过夜。
鼠伤寒沙门氏菌的继代培养:将0.5 ml 过夜培养的鼠伤寒沙门氏菌转移到 2.5 ml 新鲜的 LB 液体培养基中,并在 37°C 下孵育,同时以200 rpm摇动2 小时。
细菌的浓度的测定:使用1毫升继代培养的沙门氏菌鼠伤寒沙门氏菌到测量电Ë OD的值600与根据Nanophotometer p级(IMPLEN,NT80)并计算细菌的浓度,以该方程“1(OD 600 )= 1 × 10 6 CFU / μ升”。
 
L929上清液的制备
种子5 × 10 5 L929细胞于10cm P ETRI菜。
在补充有 10% FBS 的 RPMI 1640 培养基中,在培养箱(37°C,5% CO 2 )中培养 L929 细胞7 天。
收集上清液用移液管到一个50ml的离心管中。
离心机中以1 ,962 ×克10分钟,然后用上清转移一个吸管TE到一个新的50ml离心管中。˚F reeze在-80℃下。
 
BMDMS 的生成
牺牲小鼠并在喷洒75%的酒精IR全身。去除后腿的皮肤和毛皮,并在髋关节后切断后腿。置于 PBS 中。
除去从腿部的肌肉组织和切断从两端的胫骨和股骨(视频1 )。
 
 
视频 1. BMDM 的生成。(该视频是在做了中国科学院昆明动物研究所一个ccording从指引中国科学院昆明动物研究所Ø ñ动物护理和被批准的动物研究伦理委员会的中国科学院昆明动物研究所根据协议SMKX-20,6020 )。
 
冲洗骨髓(BM)细胞与L929上清液含有荷兰国际集团的DMEM / F-12介质(1:3)补充有10%FBS,1%非必需氨基酸和1%青霉素-链霉素使用一个5毫升注射器一个26克针。吸出冲洗骨髓几次通过20毫升注射器的18号针,以获得单细胞悬浮液。
在培养箱(37°C,5% CO 2 )中培养 BM 细胞5 天。我们Ë 10毫升介质每个P ETRI菜和5ml新鲜L929上清液含有的添加荷兰国际集团DMEM / F-12培养基中于P ETRI盘上3天。
 
细胞载玻片的制备
将高压灭菌的盖玻片放在12 孔板上。
用PBS洗涤三次,对于每次3分钟。
刮细胞从培养皿一个细胞刮刀,重悬在10毫升DMEM / F-12培养基与10%FBS ,并使用计数细胞一个细胞计数室。
将5 × 10 5 BMDM 播种到 12 孔板中,并在 37°C和5% CO 2下孵育过夜。中号确认阿克细胞融合的第二天也就是60%左右。
 
细胞刺激和固定
用500变介质μ新鲜DMEM / F-12培养基的升未经处理前L929上清液。
治疗的BMDM与不同的条件,如下小号,并在37°C孵育和5%CO 2 。
刺激的BMDM与2 μ升LPS的(500毫微克/毫升,4小时)和2 μ的ATP升(5毫米,20分钟)。
转染的BMDM与双链DNA(2 μ的升1.5 μ使用克载体对照质粒)的X-FECT转染试剂15分钟(X-FECT聚合物0.45微升和X-FECT缓冲器100微升)。
传染的BMDM with10 μ的升Francisella novicida U112 菌株(100 MOI,12 小时)。
传染的BMDM with10 μ l的沙门氏菌鼠伤寒沙门氏菌(3 MOI,1个小时)。
通过抽吸从每个孔中取出培养基,并用 PBS轻轻旋转洗涤 3 次。
通过抽吸去除 PBS,并在室温下用商业4% 多聚甲醛固定15 分钟。
              用吸尘器多聚甲醛和洗上固定的BMDM的盖玻片轻轻搅拌使其与500 µl PBS 三次。
 
免疫荧光染色
阻断和渗透细胞250-300 µl 1% BSA 和 0.1% 皂苷15-25 °C (RT) 1 小时。
通过抽吸并轻轻旋转删除块缓冲器的用PBS三次将细胞用于每次3分钟。
将细胞在 300 µl 一抗溶液(1% BSA 中的抗 ASC、0.1% 皂苷、1:150 稀释)中在 4 °C 下孵育过夜。
清洗细胞用500μl0.1%PBST三次一个上摇摆平台,对于每次3分钟。
在 15-25 °C (RT) 下,在 PBS(1:300 稀释)中用荧光偶联二抗染色细胞1 小时。
清洗细胞用500μl0.1%PBST三次一个上摇摆平台,对于每次3分钟。
用 250-300 µl DAPI(5 µg/ml,在 PBS 中制备)在 15-25 °C (RT)下染色细胞5-10 分钟。
清洗细胞用0.1%PBST三次一个上摇摆平台,对于每次3分钟。
 
盖玻片固定
取下盖片从12孔板用镊子小号和空气干燥。
将抗荧光衰减密封剂放入含有细胞的一侧。
附加的含细胞的侧盖片的至显微镜载玻片(25 × 75毫米)。滴密封剂到的边缘的盖片结合的盖玻片和显微镜载玻片在一起并防止样品干燥ING 。甲空隙气泡和空气干燥10分钟,在15-25 ℃下(RT)。
 
共聚焦扫描和图像采集
在蔡司-LSM880 共聚焦显微镜(100 倍,油浸透镜)上扫描细胞。
执行检查与ZEISS-LSM880共聚焦激光扫描显微镜使用e以下设置:激发488nm和发射530nm处为ASC斑点检测,正常的扫描速度,和帧1024 × 1024中检测到,并在图中示出的ASC斑点形成1.图像获取被执行与软件ZEN black_2-3SP1。
分析通过使用数据的软件ZEN蓝色2.6(协议在补充材料)。
 
食谱
 
TSB 固体培养基 (1 L)
30 克 BBL TM胰蛋白酶TM大豆肉汤
15 克琼脂
121°C 高压灭菌 15 分钟。
当温度在 60°C 左右时,将 L-乙酰半胱氨酸(1% 体积比)加入到高压灭菌的培养基中。
TSB 液体培养基 (1 L)
30 克 BBL TM胰蛋白酶TM大豆肉汤
121°C 高压灭菌 15 分钟。
当温度在 60°C 左右时,将 L-乙酰半胱氨酸(1% 体积比)加入到高压灭菌的培养基中。
LB 固体培养基 (1 L)
10 克氯化钠
10 克胰蛋白胨
5 克 酵母提取物
15 克琼脂
121°C 高压灭菌 15 分钟
LB 液体培养基 (1 L)
10克娜Ç升
10 克胰蛋白胨
5 克 酵母提取物
121°C 高压灭菌 15 分钟
1% BSA
1 克牛血清白蛋白
100 毫升 PBS
0.1%皂甙
0.1克皂角苷
100 毫升 PBS
0.1% PBST
1 毫升吐温® 20             
1升PBS
 
致谢
 
这项工作是由中国国家自然科学基金(31970896,31701134的支持,和81701578)。
 
利益争夺
 
作者宣称没有利益冲突。
 
伦理
 
本研究已获得中国科学院昆明动物研究所机构审查委员会的批准。 甲nimal实验在这个协议:SMKX-20 1 6020 ; 有效期:2017年1月至2022年1月。
 
参考
 
Agrawal, I. 和 Jha, S.(2020 年)。对 ASC 结构和功能在免疫稳态和疾病中的综合评价。Mol Biol Rep 47 (4) :3077-3096。
Beilharz, M.、De Nardo, D.、Latz, E. 和 Franklin, BS (2016)。测量 NLR 寡聚化 II:通过共聚焦显微镜和免疫荧光检测 ASC 斑点形成。方法 Mol Biol 1417:145-158。
Guo, Y., Li, L., Xu, T., Guo, X., Wang, C., Li, Y., Yang, Y., Yang, D., Sun, B., Zhao, X., Shao, G. 和 Qi, X. (2020)。HUWE1 介导炎症小体激活并促进宿主对细菌感染的防御。J Clin Invest 130(12): 6301-6316。
Hoss, F., Rolfes, V., Davanso, MR, Braga, TT 和 Franklin, BS (2018)。通过流式细胞术和化学交联检测 ASC 斑点形成。分子生物学方法1714:149-165。
Rathinam, VA 和 Fitzgerald, KA (2016)。炎性体复合物:新兴机制和效应器功能。单元格165(4):792-800。
Sester, DP, Thygesen, SJ, Sagulenko, V., Vajjhala, PR, Cridland, JA, Vitak, N., Chen, KW, Osborne, GW, Schroder, K. 和 Stacey, KJ (2015)。一种评估炎症小体形成的新型流式细胞术方法。J Immunol 194(1): 455-462。
Stutz, A., Horvath, GL, Monks, BG 和 Latz, E. (2013)。ASC 斑点形成作为炎症小体激活的读数。方法 Mol Biol 1040:91-101。
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引用:Li, L., Mao, R. and Qi, X. (2021). Microscopic Detection of ASC Inflammasomes in Bone Marrow Derived Macrophages Post Stimulation. Bio-protocol 11(17): e4151. DOI: 10.21769/BioProtoc.4151.
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