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Mono Sodium Urate Crystal-induced Peritonitis for in vivo Assessment of Inflammasome Activation
单钠尿酸盐晶体诱导的腹膜炎用于体内评估炎症体激活   

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参见作者原研究论文

本实验方案简略版
The Journal of Clinical Investigation
May 2016

Abstract

Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of inflammatory cells, including neutrophils from the blood into damaged/inflamed tissues. This protocol describes a method to study in vivo inflammasome activation via intraperitoneal injection of MSU crystals. MSU-injection results in a drastic increase of intraperitoneal IL-1β levels, promoting neutrophil infiltration. Early-stage neutrophil numbers correlate with the amount of released IL-1β and can be used as a read-out for the extent of in vivo inflammasome activation. In addition, this protocol might also be used as a sterile peritonitis model, to investigate mechanisms of neutrophil recruitment to the peritoneum, or as a means to obtain large numbers of in vivo activated neutrophils.

Keywords: (sterile) Peritonitis ((无菌)腹膜炎), Inflammasome (炎症体), IL-1 (IL-1), NLRP3 (NLRP3), NOD-like receptors (NOD样受体), Innate immunity (先天免疫), Neutrophil recruitment (中性粒细胞招募)

Background

Innate immune cells recognize pathogens through a set of pattern recognition receptors (PRR), which bind to evolutionarily conserved structures on the pathogen surfaces or through ligation of other danger-associated molecular patterns. One family of these receptors are the NOD-like receptors (NLR), which react to the intracellular presence of invading pathogens and/or intracellular danger signals (Meylan et al., 2006). Several PRR, including some NLRs are capable of inducing the formation of so-called inflammasome complexes, which mediate the proteolytic activation of pro-IL-1β, pro-IL-18, and other IL-1 family cytokines (Martinon et al., 2002). Due to the potent pro-inflammatory nature of IL-1β and IL-18, inflammasome activation is a highly regulated, two-step process, involving limited transcription of pro-IL-1β/pro-IL-18, and highly regulated activation of inflammasome receptors (Martinon et al., 2009). NLRP3, one of the most studied inflammasome receptors, responds to a great variety of intracellular danger-associated molecular patterns, including bacterial cell wall components (Martinon et al., 2004), damaged mitochondria (Zhou et al., 2011), and particulate materials (Martinon et al., 2006). Due to their particulate structure, mono sodium urate (MSU) crystals are very potent NLRP3 activators (Martinon et al., 2006), which are widely used for in vitro studies of NLRP3 activation.

In addition to its use for in vitro experiments, MSU can also be used to study the in vivo relevance of inflammasome activation. Here, we described an MSU-induced peritonitis model to easily and quickly study the in vivo relevance and extent NLRP3-inflammasome activation, e.g., upon genetic deletion of proteins that are involved in NLRP3 activation (Chen et al., 2006, Spalinger et al., 2016). In the MSU-induced peritonitis, the first wave of infiltrating immune cells consists mainly of neutrophils, and in the early phase of peritonitis, the number of infiltrating neutrophils correlates with the extent of inflammasome activation and with the production of mature IL-1β (Chen et al., 2006; Spalinger et al., 2016).

Materials and Reagents

  1. Pipette tips
  2. Insulin syringes (BD, catalog number: 324826 )
  3. 5 ml syringes (BD, catalog number: 302187 )
  4. 25 G needles (Terumo, catalog number: GS-351 )
  5. 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
  6. FACS tubes with lid (Corning, Falcon®, catalog number: 352058 )
  7. Mice: C57BL/6 adult females (THE JACKSON LABORATORY, catalog number: 000664 )
    Note: This protocol has been developed for C57BL/6 mice. For other mouse strains, MSU concentration and optimal time until peritoneal lavage should be titrated.
  8. Mono-sodium urate (MSU) crystals (InvivoGen, catalog number: tlrl-msu )
  9. Fluorescent antibody against Ly6G (for example, AlexaFluor647 anti-Ly6G [clone 1A8], BioLegend, catalog number: 127609 )
  10. Fluorescent antibody against Ly6B.2 (also known as 7/4 antigen; for example Fitc anti-Ly6B.2 [clone REA115], Miltenyi Biotec, catalog number: 130-103-318 )
  11. Fluorescent antibody against CD3ε (for example, PE-CF594 anti-CD3ε [clone 145-2C11], BD, BD Biosciences, catalog number: 562286 )
  12. Fluorescent antibody against CD45 (for example, Pacific Blue anti-CD45 [clone 30F11], BioLegend, catalog number: 103126 )
  13. Live-dead discriminator (for example Zombie NIR Fixable Viability Kit, BioLegend, catalog number: 423105 )
  14. Mouse IL-1 beta/IL-1F2 DuoSet ELISA kit (R&D Systems, catalog number: DY401 )
  15. Substrate Reagent Pack (R&D Systems, catalog number: DY999 ) for ELISA
  16. Dulbecco’s modified PBS (Sigma-Aldrich, catalog number: D8537-500ML )
  17. Fetal calf serum (for example, PAN-Biotech, catalog number: P40-47100 )
  18. FACS buffer (see Recipes)

Equipment

  1. Pipettes
  2. Dissection tools (sharp scissors and forceps)
  3. Neubauer cell counting chamber or automated cell counter
  4. Refrigerated benchtop centrifuge
  5. Flow cytometer
  6. ELISA plate reader

Procedure

The whole procedure is summarized in Figure 1. All animal experiments were performed in accordance to Swiss animal welfare legislation.


Figure 1. Overview of the procedure. The scheme summarizes the principal steps of this protocol for assessing in vivo inflammasome activation via peritoneal injection of MSU.

  1. MSU injection into the peritoneal cavity (see Video 1, which shows how to perform intraperitoneal injections)

    Video 1. Procedure to perform intra-peritoneal injections

    1. Prepare MSU suspension: add 0.5 ml of sterile PBS to one vial of MSU crystals (5 mg) and vortex thoroughly (> 5 min for initial resuspension, later vortex for 1 min is sufficient) to obtain a suspension of 10 mg/ml MSU.
      Note: MSU crystals do NOT dissolve in PBS and are injected as a suspension. Do not centrifuge; vortex for 1 min prior to use.
    2. Mark each mouse by ear punch or toe clipping as per local animal welfare legislation and animal experimental license.
      Note: Since the experiment lasts max 16 h, mice can also be marked transiently using a waterproof bench marker.
    3. Inject 180 μl of MSU suspension or 180 μl sterile PBS (control mice) into the peritoneal cavity using an insulin syringe:
      1. Vortex the MSU suspension before drawing into the syringe.
      2. Hold the mouse slightly inclined towards its head.
      3. Insert the needle at a 30°-45° angle. Make sure that you are in the peritoneal cavity and slowly inject the suspension.

  2. Peritoneal lavage and collection of cells
    Perform peritoneal lavage as shown in Video 2 at the desired time-point of analysis (typically 4 h, 8 h, and 16 h after MSU injection):

    Video 2. Procedure to perform a peritoneal lavage

    1. Euthanize the mouse by cervical dislocation or CO2-asphyxiation.
      Note: Process one mouse after each other, the mice should not become stiff before the cell harvest is complete. Take care that no blood vessels bleed into the peritoneal cavity when euthanizing by cervical dislocation.
    2. Open the skin of the belly carefully without damaging the peritoneum.
    3. Inject 5 ml PBS into the peritoneal cavity using a 25 G needle.
    4. Shake the mouse for 2-3 min.
    5. Aspirate the PBS from the peritoneal cavity using the same syringe, transfer into a 50 ml conical tube, measure the amount of recovered PBS.
      Note: An experienced experimenter recovers approx. 4 ml of the injected PBS.
    6. Determine the cell concentration per ml using a Neubauer counting chamber or an automated cell counter.
      Note: Red cell lysis is not required, but make sure not to count red blood cells, debris, or dead cells.

  3. Flow cytometry to characterize cell infiltrate
    1. Take 1 x 106 cells from each lavage, spin down, transfer to FACS tube.
    2. Resuspend the cells in 50 μl PBS containing:
      1. AlexaFluor anti-Ly6G antibody, 1 μg/ml;
      2. Fitc anti-Ly6B.2 antibody, 1 μg/ml;
      3. PE-CF594 anti-CD3ε antibody, 0.5 μg/ml;
      4. Pacific Blue anti-CD45 antibody, 0.5 μg/ml;
      5. Zombie-NIR Live-dead discriminator (dilute 1:800).
    3. Incubate for 20 min on ice in the dark.
    4. Add 100 μl FACS buffer to each tube.
    5. Spin down at 350 x g for 5 min.
    6. Remove supernatant and wash once more with 100 μl FACS buffer.
    7. Resuspend in 100 μl FACS buffer and proceed to analysis at Flow cytometer. Figure 2 shows typical results and gating strategy used to identify live, single, CD45+ cells.


      Figure 2. Gating strategy. Gating strategy to exclude debris, doublets, and dead cells from the analysis.

    8. CD45+, Ly6G+, Ly6B.2+ cells are neutrophils. Figure 3A shows typical flow cytometry dot plots when gated on single, live CD45+ cells.


      Figure 3. Representative data from flow cytometry. A. Representative flow cytometry plots of peritoneal cells collected 4 h after intraperitoneal injection of MSU. B. Representative results of relative and absolute numbers of infiltrating neutrophils (left) and T cells (right). Each point represents one mouse.

  4. Quantification of IL-1β in peritoneal lavage
    1. Take 0.5 ml of peritoneal lavage from Step B6.
    2. Spin the cells down, use supernatant for the analysis.
      Note: IL-1β levels can be rather low; do not dilute the supernatant for ELISA analysis.
    3. Perform IL-1β ELISA according to the manufacturer’s instructions.
      Figure 4 shows typical results of IL-1β ELISA on peritoneal lavages.


      Figure 4. Representative data from ELISA measurement. IL-1β ELISA from peritoneal lavage collected from WT and NLRP3-/- mice 8 h after intra-peritoneal MSU injection. For the lavage, 3 ml of PBS was injected into the peritoneal cavity. Each dot represents one mouse.

Data analysis

The number of infiltrating neutrophils is calculated as follows:

  1. Calculate the absolute number of neutrophils: cell concentration (cell count per ml) obtained in Step B6 x 5 = total number of cells.
  2. Calculate the number of infiltrating neutrophils: the total number of cells x frequency of Ly6G+, Ly6C+ cells of single, live, CD45+ cells = absolute number of infiltrating neutrophils.
    Accordingly, the calculation would be:

    With cell conc. = concentration of cells obtained in Step B6; and frequency = proportion of cells in the Lys6G+/Ly6B.2+ gate (see Figure 3A, left panels).
    If desired, the same approach is used to determine absolute numbers of T cells in the peritoneum, using the frequency of CD3+ cells within live, single, CD45+ cells. Figure 3B shows typical results obtained with this method.

Notes

  1. All animal experiments should be carried out according to local animal welfare legislation.
  2. The used concentration of MSU was tested for C57BL/6 mice and may require titration if other mouse strains are used.
  3. As a measure for inflammasome activation, 4 h and/or 8 h are typical time-points of analysis. However, the experiment can also be performed for up to 24 h. However, at later time-points, secondary factors influence the results and number of infiltrating cells might no longer correlate directly with inflammasome activity.
  4. IL-1β levels in the peritoneal lavage can be rather low. If IL-1β measurement is the primary read-out, lavage can be performed with as little as 2.5 to 3 ml PBS. However, this results in reduced numbers of recovered cells, since only approx. 2.0 to 2.5 ml of the injected PBS can be recovered.
  5. We typically use Nlrp3-/- mice as a negative control for MSU-induced peritonitis, since these mice are defective for the inflammasome receptor primarily involved in recognizing MSU.

Recipes

  1. FACS buffer
    Supplement Dulbecco’s PBS with 2% fetal calf serum
    Keep sterile and store at 4 °C for up to 2 months

Acknowledgments

This protocol was first described by Chen et al. (2006) and was further developed for a study by our group (Spalinger et al., 2016), which was supported by the Swiss National Science Foundation (314730-146204; CRSII3_154488/1; 310030-120312), the Zürcher Universitäts-Verein, and the Swiss Philanthropy Foundation. The authors declare no conflicts of interest or competing financial interests.

References

  1. Chen, C. J., Shi, Y., Hearn, A., Fitzgerald, K., Golenbock, D., Reed, G., Akira, S. and Rock, K. L. (2006). MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals. J Clin Invest 116(8): 2262-2271.
  2. Martinon, F., Agostini, L., Meylan, E. and Tschopp, J. (2004). Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr Biol 14(21): 1929-1934.
  3. Martinon, F., Burns, K. and Tschopp, J. (2002). The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol Cell 10(2): 417-426.
  4. Martinon, F., Mayor, A. and Tschopp, J. (2009). The inflammasomes: guardians of the body. Annu Rev Immunol 27: 229-265.
  5. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. and Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440(7081): 237-241.
  6. Meylan, E., Tschopp, J. and Karin, M. (2006). Intracellular pattern recognition receptors in the host response. Nature 442(7098): 39-44.
  7. Spalinger, M. R., Kasper, S., Gottier, C., Lang, S., Atrott, K., Vavricka, S. R., Scharl, S., Gutte, P. M., Grutter, M. G., Beer, H. D., Contassot, E., Chan, A. C., Dai, X., Rawlings, D. J., Mair, F., Becher, B., Falk, W., Fried, M., Rogler, G. and Scharl, M. (2016). NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22. J Clin Invest 126(5): 1783-1800.
  8. Zhou, R., Yazdi, A. S., Menu, P. and Tschopp, J. (2011). A role for mitochondria in NLRP3 inflammasome activation. Nature 469(7329): 221-225.

简介

由于其颗粒物质,单钠尿酸盐(MSU)晶体是NOD样受体NLRP3的有效激活剂。在激活后,NLRP3诱导炎性复合体的形成,其导致成熟IL-1β的产生和释放。生物活性IL-1β是先天性免疫应答的有效激活剂,并促进炎性细胞(包括来自血液的嗜中性粒细胞)向受损/发炎组织中的募集。该协议描述了通过腹膜内注射MSU晶体研究体内炎性体活化的方法。 MSU注射导致腹膜内IL-1β水平急剧增加,促进嗜中性粒细胞浸润。早期嗜中性粒细胞数与释放的IL-1β的量相关并且可以用作体内炎性体活化程度的读数。此外,该方案也可用作无菌性腹膜炎模型,以研究嗜中性粒细胞向腹膜募集的机制,或作为获得大量体内活化的嗜中性粒细胞的手段。

【背景】先天性免疫细胞通过一组模式识别受体(PRR)识别病原体,其与病原体表面上的进化保守结构结合或通过连接其它危险相关分子模式。这些受体的一个家族是NOD-样受体(NLR),其对细胞内入侵的病原体和/或细胞内危险信号起反应(Meylan等,2006)。包括一些NLR在内的几种PRR能够诱导形成所谓的炎症复合体,所述炎性复合体介导pro-IL-1β,pro-IL-18和其他IL-1家族细胞因子的蛋白水解活化(Martinon等人,2002)。由于IL-1β和IL-18的强烈促炎特性,炎性体激活是高度调节的两步过程,涉及pro-IL-1β/ pro-IL-18的有限转录和高度调节的活化炎性体受体(Martinon et al。,2009)。 NLRP3是研究最多的炎性体受体之一,它对多种细胞内危险相关分子模式作出反应,包括细菌细胞壁组分(Martinon等,2004),受损线粒体(周氏细胞等人,2011)和颗粒材料(Martinon等,2006)。由于它们的颗粒结构,单钠尿酸盐(MSU)晶体是非常有效的NLRP3活化剂(Martinon等,2006),其广泛用于体外研究NLRP3激活。

除用于体外实验外,MSU还可用于研究炎症小体激活的体内相关性。在这里,我们描述了MSU诱导的腹膜炎模型,以便在涉及的蛋白质的基因缺失时容易且快速地研究体内相关性和程度NLRP3-炎性体激活,例如在NLRP3活化中(Chen等人,2006,Spalinger等人,2016)。在MSU诱发的腹膜炎中,第一波浸润性免疫细胞主要由中性粒细胞组成,并且在腹膜炎的早期阶段,浸润性嗜中性粒细胞的数量与炎性体激活的程度和成熟IL-1β的产生相关(Chen ,2006; Spalinger ,2016)。

关键字:(无菌)腹膜炎, 炎症体, IL-1, NLRP3, NOD样受体, 先天免疫, 中性粒细胞招募

材料和试剂

  1. 移液器吸头
  2. 胰岛素注射器(BD,目录号:324826)
  3. 5毫升注射器(BD,目录号:302187)
  4. 25 G针(Terumo,产品目录号:GS-351)
  5. 50毫升试管(Corning,Falcon ,目录号:352070)
  6. 带有盖子的FACS管(Corning,Falcon ,目录号:352058)
  7. 小鼠:C57BL / 6成年女性(THE JACKSON LABORATORY,目录号:000664)
    注:此协议已针对C57BL / 6小鼠开发。对于其他小鼠品系,应调整MSU浓度和直至腹膜灌洗的最佳时间。
  8. 单钠尿酸盐(MSU)晶体(InvivoGen,目录号:tlrl-msu)
  9. 针对Ly6G的荧光抗体(例如,AlexaFluor647抗Ly6G [克隆1A8],BioLegend,目录号:127609)
  10. 针对Ly6B.2的荧光抗体(也称为7/4抗原;例如Fitc抗Ly6B.2 [克隆REA115],Miltenyi Biotec,目录号:130-103-318)
  11. 针对CD3ε的荧光抗体(例如,PE-CF594抗CD3ε[克隆145-2C11],BD,BD Biosciences,目录号:562286)
  12. 针对CD45的荧光抗体(例如,Pacific Blue抗CD45 [克隆30F11],BioLegend,目录号:103126)
  13. 活死亡鉴别器(例如Zombie NIR Fixable Viability Kit,BioLegend,产品目录号:423105)
  14. 小鼠IL-1β/ IL-1F2 DuoSet ELISA试剂盒(R& D Systems,目录号:DY401)
  15. 用于ELISA的底物试剂包(R& D Systems,目录号:DY999)
  16. Dulbecco改良的PBS(Sigma-Aldrich,目录号:D8537-500ML)
  17. 胎牛血清(例如,PAN-Biotech,目录号:P40-47100)
  18. FACS缓冲液(见食谱)

设备

  1. 移液器
  2. 解剖工具(锋利的剪刀和镊子)
  3. Neubauer细胞计数室或自动细胞计数器
  4. 冷冻台式离心机
  5. 流式细胞仪
  6. ELISA平板阅读器

程序

整个过程总结在图1中。所有的动物实验均按照瑞士动物福利法进行。


图1.程序概述该方案总结了该方案的主要步骤,用于通过腹膜内注射MSU评估体内炎性体活化。

  1. MSU注射到腹膜腔内(见视频1,该图显示了如何进行腹腔内注射)

    视频1

    1. 准备MSU悬浮液:向一小瓶MSU晶体(5mg)中加入0.5ml无菌PBS并彻底涡旋(> 5分钟用于初始重悬,稍后涡旋1分钟就足够了)以获得10mg / ml MSU的悬浮液。
      注意:MSU晶体不溶于PBS并以悬浮液形式注入。不要离心;使用前涡旋1分钟。
    2. 根据当地的动物福利法和动物实验许可证,通过耳朵或脚趾夹子标记每只小鼠。
      注意:由于实验持续时间最长为16小时,因此可以使用防水台标记暂时标记小鼠。
    3. 使用胰岛素注射器将180μlMSU悬液或180μl无菌PBS(对照小鼠)注入腹腔:

      1. 在吸入注射器之前旋转MSU悬架

      2. 按住鼠标稍微向其头部倾斜
      3. 以30°-45°的角度插入针。确保你在腹腔内,并缓慢注入悬浮液。

  2. 腹腔灌洗和收集细胞
    在所需的分析时间点(通常在MSU注射后4小时,8小时和16小时)进行视频2所示的腹膜灌洗:

    视频2


    1. 颈椎脱臼或CO 2窒息安乐死小鼠。
      注意:处理一只小鼠后,小鼠在细胞采集完成前不应变硬。请注意,颈椎脱位安乐死时血管不会渗入腹腔。

    2. 小心打开腹部皮肤,不会损伤腹膜。

    3. 使用25 G针头将5 ml PBS注入腹腔
    4. 摇动鼠标2-3分钟。
    5. 使用相同的注射器从腹膜腔内吸出PBS,转移到50ml锥形管中,测量回收的PBS的量。
      注意:有经验的实验者可以恢复约。 4毫升注射的PBS。
    6. 使用Neubauer计数室或自动细胞计数器确定每毫升的细胞浓度。
      注:红细胞裂解不是必需的,但要确保不要计数红细胞,碎片或脱细胞。

  3. 流式细胞术来表征细胞浸润
    1. 从每个灌洗液中取1×10 6个细胞,旋下,转移到FACS管中。
    2. 重悬细胞在50μLPBS含有:

      1. AlexaFluor抗Ly6G抗体,1μg/ ml;
      2. Fitc抗Ly6B.2抗体,1μg/ ml;

      3. PE-CF594抗CD3ε抗体,0.5μg/ ml;

      4. 太平洋蓝抗CD45抗体,0.5μg/ ml;
      5. 僵尸-NIR活死查杀器(稀释1:800)。

    3. 在冰上孵育20分钟

    4. 加入100μlFACS缓冲液

    5. 在350 em xg 下旋转5分钟
    6. 取出上清液并用100μlFACS缓冲液再次清洗。
    7. 重悬于100μlFACS缓冲液中并在流式细胞仪上进行分析。图2显示了用于鉴定活的单个CD45 +细胞的典型结果和门控策略。


      图2.门控策略门控策略,用于排除分析中的碎片,双合和死亡单元。

    8. CD45 +,Ly6G +,Ly6B.2 +细胞是嗜中性粒细胞。图3A显示了当在单个活CD45 +细胞上选通时的典型流式细胞计点图。


      图3.来自流式细胞术的代表性数据A.腹膜内注射MSU后4小时收集的腹膜细胞的代表性流式细胞术图。 B.浸润性嗜中性粒细胞(左)和T细胞(右)的相对和绝对数量的代表性结果。每个点代表一只老鼠。

  4. 腹腔灌洗液中IL-1β的定量

    1. 从步骤B6中取0.5 ml腹腔冲洗液。
    2. 旋转细胞,使用上清液进行分析。
      注:IL-1β水平可能相当低;不要稀释上清液进行ELISA分析。
    3. 根据制造商的说明进行IL-1βELISA。
      图4显示了腹腔灌洗液中IL-1βELISA的典型结果。


      图4.来自ELISA测量的代表性数据。 IL-1β腹膜内MSU注射后8小时从WT和NLRP3 - / - 小鼠收集腹膜灌洗的ELISA。对于灌洗,将3ml的PBS注射到腹腔中。每个点代表一个鼠标。

数据分析

浸润性嗜中性粒细胞的数量计算如下:

  1. 计算嗜中性粒细胞的绝对数量:步骤B6中获得的细胞浓度(每毫升细胞数)×5 =细胞总数。
  2. 计算单个活的CD45 +细胞的Ly6G + + Ly6C + +细胞的细胞总数×频率计算浸润性嗜中性粒细胞的数量=渗透性嗜中性粒细胞的绝对数量。
    因此,计算将是:

    随着细胞浓度。 =步骤B6中获得的细胞浓度;和频率= Lys6G + + / Ly6B.2 + +门中细胞的比例(见图3A,左图)。
    如果需要,使用相同的方法来确定腹膜中T细胞的绝对数量,使用活的单个CD45 +细胞内CD3 +细胞的频率。图3B显示了用这种方法获得的典型结果。

笔记

  1. 所有动物实验应根据当地动物福利立法进行。
  2. 对于C57BL / 6小鼠测试MSU的使用浓度,如果使用其他小鼠品系,则可能需要滴定。
  3. 作为炎症小体激活的一种测量,4小时和/或8小时是典型的分析时间点。但是,该实验也可以进行长达24小时。然而,在较晚的时间点,次要因素影响结果,浸润细胞的数量可能不再与炎性体活性直接相关。
  4. 腹膜灌洗液中的IL-1β水平可能相当低。如果IL-1β测量是主要读数,则可以用少至2.5至3ml的PBS进行灌洗。但是,这会导致回收电池的数量减少,因为只有约。
    可以回收2.0至2.5毫升注入的PBS。
  5. 我们通常使用Nlrp3 - / - 小鼠作为MSU诱导的腹膜炎的阴性对照,因为这些小鼠对主要参与识别MSU的炎性体受体有缺陷。

食谱

  1. FACS缓冲液
    补充Dulbecco PBS和2%胎牛血清
    保持无菌状态并在4°C储存长达2个月

致谢

该协议首先由Chen等人描述。 (2006),并且由我们小组进行了进一步研究(Spalinger等人,2016年),该研究得到了瑞士国家科学基金会(314730-146204; CRSII3_154488 / 1 ; 310030-120312),ZürcherUniversitäts-Verein和瑞士慈善基金会。作者声明不存在利益冲突或竞争财务利益。

参考

  1. Chen,C.J.,Shi,Y.,Hearn,A.,Fitzgerald,K.,Golenbock,D.,Reed,G.,Akira,S。和Rock,K.L。(2006)。 MyD88依赖性IL-1受体信号传导对于由单钠盐尿酸盐晶体刺激的痛风炎症是必不可少的。 a> J Clin Invest 116(8):2262-2271。
  2. Martinon,F.,Agostini,L.,Meylan,E.和Tschopp,J。(2004)。 鉴定细菌胞壁酰二肽作为NALP3 / cryopyrin炎症小体的激活剂 Curr Biol 14(21):1929-1934。
  3. Martinon,F.,Burns,K。和Tschopp,J。(2002)。 炎症小体:触发炎性半胱天冬酶活化和proIL-β加工的分子平台。 Mol Cell 10(2):417-426。
  4. Martinon,F.,Mayor,A.和Tschopp,J。(2009)。 炎症小体:身体的监护人。 Annu Rev Immunol 27:229-265。
  5. Martinon,F.,Petrilli,V.,Mayor,A.,Tardivel,A.和Tschopp,J。(2006)。 痛风相关的尿酸晶体可激活NALP3炎性体。 440(7081):237-241。
  6. Meylan,E.,Tschopp,J.和Karin,M。(2006)。 宿主反应中的细胞内模式识别受体 Nature 442(7098):39-44。
  7. Spalinger,MR,Kasper,S.,Gottier,C.,Lang,S.,Atrott,K.,Vavricka,SR,Scharl,S.,Gutte,PM,Grutter,MG,Beer,HD,Contassot,E., Chan,AC,Dai,X.,Rawlings,DJ,Mair,F.,Becher,B.,Falk,W.,Fried,M.,Rogler,G。和Scharl,M.(2016)。 NLRP3酪氨酸磷酸化受蛋白酪氨酸磷酸酶PTPN22控制。
  8. Zhou,R.,Yazdi,A. S.,Menu,P.和Tschopp,J。(2011)。 线粒体在NLRP3炎症小体激活中的作用 Nature 469(7329):221-225。
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引用:Spalinger, M. R. and Scharl, M. (2018). Mono Sodium Urate Crystal-induced Peritonitis for in vivo Assessment of Inflammasome Activation. Bio-protocol 8(5): e2754. DOI: 10.21769/BioProtoc.2754.
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