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Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and activates IL-1β, IL-18. Examples of inflammasome forming NLRs are NLRP3, NLRP1, NAIP and NLRC4. We can easily identify new inflammasome activators by performing the following protocol.

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In vitro Inflammasome Assay
体外炎性体试验

免疫学 > 免疫细胞功能 > 细胞因子
作者: Mario M. Zaiss
Mario M. ZaissAffiliation: School of Life Sciences - Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
For correspondence: mario.zaiss@epfl.ch
Bio-protocol author page: a1389
 and Kendle M. Maslowski
Kendle M. MaslowskiAffiliation: Laboratory for Intestinal Ecosystem, RCAI RIKEN Center for Integrative Medical Sciences (IMS-RCAI), Yokohama, Japan
Bio-protocol author page: a1390
Vol 4, Iss 11, 6/5/2014, 4232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1142

[Abstract] Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and activates IL-1β, IL-18. Examples of inflammasome forming NLRs are NLRP3, NLRP1, NAIP and NLRC4. We can easily identify new inflammasome activators by performing the following protocol.

[Abstract] 先天免疫细胞通过一系列先天免疫模式识别受体(PRR)感知病原体和危险相关分子模式(PAMP和DAMP)。 一种类型的PRR是Nod样受体(NLR),其形成炎症体; 募集和激活Caspase-1所需的分子平台,其反过来切割和激活IL-1β,IL-18。 形成NLR的炎性体的实例是NLRP3,NLRP1,NAIP和NLRC4。 我们可以通过执行以下协议轻松识别新的inflammasome激活剂。

Materials and Reagents

  1. Mice (e.g. C57B/6)
  2. DMEM (Life Technologies, Gibco®, catalog number: 10566-024 )
  3. 1% Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Gibco®, catalog number: 15140148 )
  4. 1x PBS
  5. Accutase (PAA Laboratories GmbH, catalog number: L11-007 )
  6. Ultrapure Escherichia coli (E.coli) K12 LPS (Life Technologies, InvitrogenTM, catalog number: tlrl-peklps )
  7. IL-1β ELISA (eBioscience, catalog number: 88-7013-88 )
  8. Primary antibodies against IL-1β (e.g. R&D System, catalog number: AF-401-NA ) and Caspase-1 (e.g. Aidpogen International, catalog number: AG-20B-0042-C100 ) (suitable for western blot)
  9. HRP-conjugated secondary antibody (e.g. Cell Signaling anti-mouse HRP, Cell Signaling Technology, catalog number: 7076 )
  10. Nitrocellulose membrane (0.45 µm) (GE Healthcare, Hybond, catalog number: 95038-402 )
  11. 5% sodium azide in water (Sigma-Aldrich, catalog number: 26628-22-8 )
  12. Skim milk powder (Sigma-Aldrich, or your local grocer)
  13. ECL solution (Pierce, catalog number: 34095 or GE Healthcare, catalog number: RPN2133 )
  14. 1 M dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  15. Stimulus: e.g. 1 M ATP (Sigma-Aldrich, catalog number: A26209 ), 10 mM nigericin (Sigma-Aldrich, catalog number: N7143 ), 300 µg/ml monosodium urate (Michigan State University, catalog number: U2875 )
  16. Flushing medium (see Recipes)
  17. Red blood cell lysis solution (see Recipes)
  18. Bone-marrow-derived macrophage (BMM) culture medium (see Recipes)
  19. Ponceau staining solution (see Recipes)
  20. 3x western blot sample buffer (see Recipes)
  21. Blocking buffer (see Recipes)
  22. Running buffer (see Recipes)
  23. Blotting buffer (see Recipes)

Equipment

  1. Razor blade
  2. Laminar flow hood
  3. Bench top centrifuge with 96-well plate adaptors
  4. 10 ml syringe
  5. 22 G gauge needles
  6. 18 G blunt needles
  7. 100-µm cells strainers
  8. 10 cm cell-culture treated petri dishes
  9. MaxiSorb ELISA plates (Nunc®)
  10. 96-well tissue culture plate
  11. ELISA plate reader
  12. Western blot equipment (protean mini 1 mm) (Bio-Rad Laboratories)
  13. Film, developer, dark room or equivalent development equipment
  14. Parafilm or plastic

Software

  1. ELISA analysis software

Procedure

  1. Bone marrow isolation and cell culture
    1. Sacrifice mice by CO2 inhalation and cervical dislocation.
    2. Remove the fibia and tibia and place in flushing medium on ice.
    3. Under a laminar flow hood clean bones, removing excess muscle by scraping with razor blade, and cut ends of bones.
    4. Flush bones with flushing medium, using a 10 ml syringe and a fine-gauge needle.
    5. Resuspend clumps from bone by passing through the syringe with a 18 G blunt needle.
    6. Filter through 100 µM cell strainer.
    7. Pellet cells (5 min at 1,200 rpm).
    8. Resuspend in 1 to 5 ml RBC lysis buffer, incubate for 5 min at room temperature.
    9. Add 14 ml PBS, pellet cells (5 min at 1,200 rpm).
    10. Count and wash with PBS.
    11. Resuspend cells in BMM medium at a density of 106/ml, plate 10 ml per 10 cm Petri dish.
    12. Culture cells for 6 to 7 days at 37 °C, 5% CO2, topping up medium on day 3 and 6 with 5 ml BMM medium.
    13. After 6-7 days remove the culture supernatant, wash petri dish once with 5 ml PBS. Adherent macrophages appear flat, with round cell body and extending dendrites. Flow cytometry analysis for macrophage markers, such as F480/CD11b can be used to determine macrophage purity
    14. Remove adherent BMMs with 5 ml accutase, leaving petri dishes to incubate at room temperature until cells become rounded and start to lift (around 10 min).
    15. Wash cells with PBS.
    16. Count and resuspend in BMM medium at 106/ml, and plate 200 µl/well in a flat-bottom 96-well tissue culture plate.
    17. Incubate overnight at 37 °C to enable cells to become adherent.

  2. Stimulation
    1. Prime cells: remove cell supernatant and add 200 µl fresh BMM medium with 20 ng/ml ultrapure LPS, incubate at 37 °C for 3 h. For a negative control add medium without LPS. BMMs derived from Caspase1/11-deficient mice can also be used as a negative control.
      Note: BMMs require a priming step in order to induce expression of inflammasome components, such as Nlrp3 (Some stimuli may activate NF-κB pathway and induce priming without the need for LPS-priming.).
    2. Add stimuli to cells in 50 µl BMM medium, without removing medium with LPS (You can also remove medium with LPS if desired.).
    3. Incubate at 37 °C, time depending on the stimulus, e.g. 1 M ATP or 10 mM nigericin for 30 min to 1 h, 300 µg/ml MSU for 6 h.
      Note: For Helminth inflammasome activation we used HES (5 or 50 μg/ml), pyrogen-free HES (P.HES) (5 or 50 μg/ml) or homogenized Heligmosomoides polygyru (H. polygyrus) L5 parasite (HPL5) (100 μg/ml), and incubated overnight.
    4. Spin down the plate (1,400 rpm for 3 min), collect the cell supernatants for ELISA or WB analysis, freeze until use.
      Note: Aim to collect about 20 -50 μl less than the initial volume you put in.
    5. Wash cells 1x with cold PBS and add 40 µl 1x western blot sample buffer supplemented with fresh 10% 1 M DTT directly to cells (can freeze until use or proceed to preparation for WB, below).

  3. Measurement
    1. Perform IL-1β ELISA with 50 - 100 µl of cell supernatant as per manufacturers' instructions.
    2. Proceed to western blot with samples you want to test further.
    3. For cell extracts, pool each triplicate into 1 tube.
    4. For cell supernatant, take an aliquot of supernatant and add to 2 parts of 3x western blot sample buffer (with 10% fresh 1 M DTT). Load 20 to 30 μl of the supernatant.
    5. Option: concentrate protein from the supernatant:
      1. Add equal volumes of supernatant and methanol, and add chloroform 1/8 of the volume of supernatant, mix well. (e.g. 100 μl supernatant + 100 μl  methanol + 12.5 μl chloroform)
      2. Centrifuge for 3 min at top speed (in bench-top eppendorf centrifuge).
      3. Remove as much liquid as possible without disturbing the pellet at the interface.
      4. Add another volume of methanol (same as initial), mix well.
      5. Centrifuge at top speed for 3 min.
      6. Remove supernatant, careful not to disrupt protein pellet.
      7. Air dry for approx. 20 min.
      8. Add 20 to 40 µl 1x western blot sample buffer (with 10% fresh 1 M DTT), depending on how concentrated you want it.
    6. Boil samples at 95 °C for 5 min, then cool on ice.
    7. Subject 20-30 µl sample to polyacrylamide gel electrophoresis using a 15% gel and running buffer.
    8. Blot the proteins onto a nitrocellulose membrane using the blot buffer.
    9. To ensure loading was even, stain membrane in ponceau red solution for approx. 2 min, wash with distilled water until excess stain is removed, make a copy of the blot for your documentation.
    10. Block the membrane by incubating in blocking buffer for at least 5 min at room temperature with mild shaking.
    11. Add your antibody, anti-IL-1β or caspase-1 (which detect pro and cleaved forms) (1:1,000 to 1:2,000 in blocking buffer containing 0.05% azide) and incubate overnight at 4 °C with mild shaking (keep antibody source at -20 °C for repeated use).
    12. Wash 3 x 5 min with PBS-tween (0.5%) at room temperature with mild shaking.
    13. Add HRP-conjugated secondary antibody 1:5,000-1:10,000 in blocking buffer and incubate for 1 h at room temperature with mild shaking.
    14. Wash for at least 20 min in PBS-tween (changing the buffer at least 4 times) at room temperature with mild shaking.
    15. Dry the membrane on a tissue, immediately lay it over a 1-ml drop of regular or high fidelity ECL solution (on a piece of parafilm or plastic) for 1 min, remove excess solution on a tissue.
    16. Develop the blot using standard techniques and equipment (Detection of cleaved IL-1β and caspase-1 in the cell supernatant indicates inflammasome activation.).
    17. Repeat steps 9-15 with additional antibodies (e.g. tubulin for loading control).

Recipes

  1. Flushing medium
    DMEM
    1% Penicillin-Streptomycin
  2. Red blood cell lysis solution
    155 mM NH4Cl
    10 mM KHCO3
    1 mM EDTA
  3. BMM culture medium
    DMEM
    10% FCS
    20% supernatant from L929 cell culture
    1% Penicillin-Streptomycin
  4. 3x western blot sample buffer
    187.5 mM Tris-HCl (pH 6.8)
    6% w/v SDS
    0.03% w/v phenol red
    30% w/v glycerol (adjust to pH 6.8)
  5. Ponceau staining solution (for 500 ml)
    0.05 % Ponceau S 250 mg
    3% tricholacetic acid 15 g
  6. Blocking buffer
    5% skim milk in PBS-tween
  7. Running buffer (for 5 L)
    Tris base 75 g
    Glycine 360 g
    SDS (20%) 125 ml
  8. Blotting buffer (for 20 L)
    Tris base 50 g
    Glycine 238 g
    Ethanol 3.3 L

Acknowledgments

This protocol is an extended version of the one described in Zaiss et al. (2013). The research has received funding from the European community seventh framework program [FP7/2009–2014] under EC-GA no[241642] and part of this work was funded by grants from the Swiss National Science Foundation and the Institute of Arthritis Research. KMM is supported by EMBO long-term fellowship and the Australian NHMRC post-doctoral fellowship.

References

  1. Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.

材料和试剂

  1. 小鼠(例如 C57B/6)
  2. DMEM(Life Technologies,Gibco ,目录号:10566-024)
  3. 1%青霉素 - 链霉素(10,000U/ml)(Life Technologies,Gibco ,目录号:15140148)
  4. 1x PBS
  5. Accutase(PAA Laboratories GmbH,目录号:L11-007)
  6. 超纯大肠杆菌(大肠杆菌)K12 LPS(Life Technologies,Invitrogen TM ,目录号:tlrl-peklps)
  7. IL-1βELISA(eBioscience,目录号:88-7013-88)
  8. 针对IL-1β的初级抗体(例如R& D系统,目录号:AF-401-NA)和胱天蛋白酶-1(例如Aidpogen International,目录号:AG- 20B-0042-C100)(适用于western印迹)
  9. HRP-结合的二抗(例如,Cell Signaling抗小鼠HRP,Cell Signaling Technology,目录号:7076)。
  10. 硝化纤维素膜(0.45μm)(GE Healthcare,Hybond,目录号:95038-402)
  11. 5%叠氮化钠水溶液(Sigma-Aldrich,目录号:26628-22-8)
  12. 脱脂奶粉(Sigma-Aldrich,或当地杂货店)
  13. ECL溶液(Pierce,目录号:34095或GE Healthcare,目录号:RPN2133)
  14. 1 M二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  15. 刺激:例如1M ATP(Sigma-Aldrich,目录号:A26209),10mM尼日利亚菌素(Sigma-Aldrich,目录号:N7143),300μg/ml尿酸单钠(密歇根州立大学目录 号码:U2875)
  16. 冲洗介质(见配方)
  17. 红细胞裂解液(见配方)
  18. 骨髓来源的巨噬细胞(BMM)培养基(见Recipes)
  19. Ponceau染色溶液(参见配方)
  20. 3x western印迹样品缓冲液(见配方)
  21. 阻止缓冲区(参见配方)
  22. 运行缓冲区(参见配方)
  23. 印迹缓冲液(参见配方)

设备

  1. 剃刀刀片
  2. 层流罩
  3. 带96孔板适配器的台式离心机
  4. 10毫升注射器
  5. 22 G号针
  6. 18 G钝针
  7. 100μm细胞滤网
  8. 10cm细胞培养处理的培养皿
  9. MaxiSorb ELISA板(Nunc )
  10. 96孔组织培养板
  11. ELISA酶标仪
  12. Western印迹设备(Protean mini 1mm)(Bio-Rad Laboratories)
  13. 电影,开发商,暗室或同等的开发设备
  14. 石蜡膜或塑料

软件

  1. ELISA分析软件

程序

  1. 骨髓分离和细胞培养
    1. 牺牲小鼠CO 2吸入和颈脱臼
    2. 取出腓骨和胫骨,置于冰上的冲洗介质中。
    3. 在层流罩下清洁骨头,通过用刀片刮削去除多余的肌肉,并切除骨头。
    4. 用冲洗介质冲洗骨头,使用10 ml注射器和细针头
    5. 通过用18 G钝针穿过注射器重悬从骨头团块
    6. 骨髓分离和细胞培养
      1. 牺牲小鼠CO 2吸入和颈脱臼
      2. 取出腓骨和胫骨,置于冰上的冲洗介质中。
      3. 在层流罩下清洁骨头,通过用刀片刮削去除多余的肌肉,并切除骨头。
      4. 用冲洗介质冲洗骨头,使用10 ml注射器和细针头
      5. 通过用18 G钝针穿过注射器重悬从骨头团块
      6. ...
      7. After 6-7 days remove the culture supernatant, wash petri dish once with 5 ml PBS. Adherent macrophages appear flat, with round cell body and extending dendrites. Flow cytometry analysis for macrophage markers, such as F480/CD11b can be used to determine macrophage purity
      8. Remove adherent BMMs with 5 ml accutase, leaving petri dishes to incubate at room temperature until cells become rounded and start to lift (around 10 min).
      9. Wash cells with PBS.
      10. Count and resuspend in BMM medium at 106/ml, and plate 200 µl/well in a flat-bottom 96-well tissue culture plate.
      11. Incubate overnight at 37 °C to enable cells to become adherent.

    7. Stimulation
      1. Prime cells: remove cell supernatant and add 200 µl fresh BMM medium with 20 ng/ml ultrapure LPS, incubate at 37 °C for 3 h. For a negative control add medium without LPS. BMMs derived from Caspase1/11-deficient mice can also be used as a negative control.
        Note: BMMs require a priming step in order to induce expression of inflammasome components, such as Nlrp3 (Some stimuli may activate NF-κB pathway and induce priming without the need for LPS-priming.).
      2. 添加刺激 细胞在50μlBMM培养基中,不用LPS去除培养基(你可以 如果需要,也用LPS除去培养基。
      3. 在37℃孵育, 时间取决于刺激,例如1M ATP或10mM尼日利亚霉素30分钟 min至1h,300μg/ml MSU 6小时。
        注意:对于Helminth inflammasome激活   我们使用HES(5或50μg/ml),无热原HES(P.HES)(5或50μg/ml) 或匀浆的 ( H。polygyrus )L5寄生虫(HPL5)(100μg/ 孵育过夜。
      4. 旋转板(1,400 rpm 3分钟),收集细胞上清液用于ELISA或WB分析,冷冻,直到使用。
        注意:目标是收集比您投入的初始音量少大约20-50μl。
      5. 用冷PBS洗涤细胞1次,加入40μl1×Western blot 缓冲液中直接添加新鲜10%1M DTT至细胞(可冷冻   直到使用或进行WB的制备,下面)。

    8. 测量
      1. 根据制造商的说明书,用50 - 100μl的细胞上清液进行IL-1βELISA。
      2. 用您想要进一步测试的样品进行免疫印迹
      3. 对于细胞提取物,将每三份重复混合到1个试管中
      4. 对于细胞上清液,取上清液的等分试样并加至2 部分的3x蛋白质印迹样品缓冲液(含10%新鲜1M DTT)。 负载20   至30μl的上清液
      5. 选项:从上清液浓缩蛋白质:
        1. 加入等体积的上清液和甲醇,加入氯仿1/8 的上清液,混匀。 (例如100μl上清液+ 100 μl  甲醇+12.5μl氯仿)
        2. 以最高速度离心3分钟(在台式eppendorf离心机中)
        3. 尽可能多地去除液体,而不会在界面处干扰沉淀
        4. 加入另一体积的甲醇(与初始体积相同),混匀
        5. 以最高速度离心3分钟。
        6. 除去上清液,小心不要破坏蛋白质沉淀。
        7. 空气干燥约。 20分钟。
        8. 加入20至40μl1 x Western blot样品缓冲液(含10%新鲜1M DTT),取决于您想要的浓度。
      6. 在95℃下煮沸样品5分钟,然后在冰上冷却
      7. 主题20-30μl样品使用15%凝胶和运行缓冲液进行聚丙烯酰胺凝胶电泳
      8. 使用印迹缓冲液将蛋白质印迹到硝酸纤维素膜上
      9. 为了确保加载均匀,在ponceau红色溶液中染色膜   约。 2分钟,用蒸馏水洗涤直至除去多余的污渍,   制作文档的印迹的副本。
      10. 通过在封闭缓冲液中室温孵育至少5分钟,轻轻摇动封闭膜
      11. 添加您的抗体,抗IL-1β或caspase-1(其检测pro和 裂解形式)(1:1,000至1:2,000,在含有0.05% 叠氮化物),并在4℃下温和摇动(保持抗体)孵育过夜 源在-20°C重复使用)。
      12. 在室温下轻轻摇动用PBS-吐温(0.5%)洗涤3×5分钟
      13. 在封闭中加入HRP-结合的二抗1:5,000-1:10,000 缓冲液中,并在室温下温和摇动孵育1小时
      14. 在室温下轻轻摇动,在PBS-tween中至少洗涤20分钟(更换缓冲液至少4次)。
      15. 干燥组织上的膜,立即将其置于1-ml滴上 定期或高保真ECL溶液(在一片石蜡膜上或 塑料)1分钟,去除多余的溶液在组织上
      16. 使用标准技术和设备(检测 裂解的IL-1β和caspase-1在细胞上清液中的表达 炎症小体激活。)。
      17. 重复步骤9-15与其他抗体(例如微管蛋白加载控制)。

    食谱

    1. 冲洗介质
      DMEM
      1%青霉素 - 链霉素
    2. 红细胞裂解液
      155mM NH 4 Cl/v 10mM KHCO 3
      1mM EDTA
    3. BMM培养基
      DMEM
      10%FCS
      来自L929细胞培养物的20%上清液 1%青霉素 - 链霉素
    4. 3x western印迹样品缓冲液
      187.5mM Tris-HCl(pH6.8) 6%w/v SDS
      0.03%w/v酚红
      30%w/v甘油(调节至pH 6.8)
    5. Ponceau染色溶液(500ml) 0.05%Ponceau S 250mg
      3%三氯乙酸15g
    6. 阻塞缓冲区
      PBS-tween中的5%脱脂牛奶
    7. 运行缓冲液(5 L)
      Tris碱75 g
      甘氨酸360 g
      SDS(20%)125ml
    8. 印迹缓冲液(20 L)
      Tris碱50μg
      甘氨酸238 g
      乙醇3.3L

    致谢

    该协议是Zaiss等人(2013)中描述的扩展版本。 研究获得了欧洲共同体第七框架计划[FP7/2009-2014]在EC-GA no [241642]下的资助,这项工作的一部分由瑞士国家科学基金会和关节炎研究所资助。 KMM由EMBO长期研究金和澳大利亚NHMRC博士后研究金支持。

    参考文献

    1. Zaiss,M.M.,Maslowski,K.M.,Mosconi,I.,Guenat,N.,Marsland,B.J.and Harris,N.L。(2013)。 IL-1beta抑制先天性IL-25和IL-33的产生,并维持蠕虫的长期性。 PLoS Pathog 9(8):e1003531。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zaiss, M. M. and Maslowski, K. M. (2014). In vitro Inflammasome Assay. Bio-protocol 4(11): e1142. DOI: 10.21769/BioProtoc.1142; Full Text
  2. Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.




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Kendle M. Maslowski的其他实验方案(1)