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Isolation of Particles of Recombinant ASC and NLRP3
重组表达的ASC 和NLRP3颗粒的分离   

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Abstract

NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) (Schroder and Tschopp, 2010). We and others have reported that upon inflammasome activation functional oligomeric inflammasome particles of NLRP3 and ASC were released from cells, acting as danger signals to amplify inflammation by promoting the activation of caspase-1 extracellularly (Baroja-Mazo et al., 2014; Franklin et al., 2014).
Studying the extracellular function of oligomeric ASC and NLRP3 inflammasome particles was possible by purification of recombinant particles of ASC or the constitutively activated NLRP3 mutant associated with cryopyrin-associated periodic syndromes (CAPS, mutation p.D303N), both tagged with the yellow fluorescent protein (YFP) and expressed in HEK293 cells. The purification process was facilitated by the fact that expression of recombinant ASC or mutant NLRP3 in HEK293 cells resulted in their spontaneous aggregation into specks (Baroja-Mazo et al., 2014) and the protocol was originally adapted from Fernandes-Alnemri and Alnemri (2008).

Materials and Reagents

  1. HEK293T cell line (ATCC® number: CRL-11268 )
  2. Dulbecco´s Modified Eagle´s medium F12 (DMEM-F12) (Biowest, catalog number: L0090 )
  3. 10% (v/v) Fetal Bovine Serum (Lonza, catalog number: DE14-801F )
  4. 200 mM L-Glutamine (Lonza, catalog number: 17-605E )
  5. Penicillin and streptomycin (Lonza, catalog number: 17-603E )
  6. Liquid nitrogen
  7. 1x DPBS (Life Technologies, Gibco®, catalog number: 14190-094 )
  8. 1x Percoll (see Recipes)
  9. Buffer A (see Recipes)
  10. 2x CHAPS (see Recipes)
  11. 1x CHAPS (see Recipes)

Equipment

  1. Tissue culture flasks with quick-release screw cap 75 ventilation (SARSTEDT AG, catalog number: 83.1813.002 )
  2. Tissue culture plate with lid, hydrophobic, sterile (SARSTEDT AG, catalog number: 83.1839 )
  3. 37 °C, 5% CO2 cell culture incubator
  4. Table top cooling centrifuge with rotor for 15 ml Falcon tubes (Sigma-Aldrich, catalog number: 3K30 )
  5. Table top cooling microcentrifuge with rotor for 2 ml tubes (HERMLE, catalog number: Z216MK )
  6. Water bath at 37 °C
  7. Syringe (1 ml) (Nipro Syringe, catalog number: SY31SCTU EC )
  8. 20 G and 25 G needles (Becton, Dickinson and Company, catalog number: 300600 )
  9. Tissue culture class II laminar flow hood (Telstar Bio II)
  10. Inverted microscope with epifluorescence (Nikon Eclipse Ti)
  11. Bürker counting chamber
  12. Ultrafree-CL low-binding Durapore PVDF membrane (5 µm) (EMD Millipore, catalog number: UFC40S25 )

Procedure

Perform all the steps on ice and all the centrifugations at 4 °C unless noted otherwise.
Start the purification from 107 HEK293 transiently expressing ASC-YFP or stably expressing NLRP3 (p.D303N)-YFP maintained in DMEM: F12 (1:1) supplemented with 10% FCS, 2 mM Glutamax and 1% penicillin-streptomycin.

  1. Wash the cells with warm PBS and detached them with 6 ml of cold PBS by pipetting up and down against the bottom of the flask. Transfer the cell suspension into a 15 ml tube (representative scheme of steps from 1 to 8 is shown in Figure 1).
  2. The cells were pelleted by centrifugation at 2,000 x g for 3 min. Discard supernatant and re-suspended the pellet in 600 µl of cold Buffer-A and transfer it into a 1.5 ml tube.
  3. Cells in buffer A were lysed on ice by passing through a 1 ml syringe with a 20 G needle (10 times) and then through a 25 G needle (20 times). After that, the lysate was freeze in liquid nitrogen (keep in liquid nitrogen around 30-40 sec or until the lysate is frozen) and subsequent thawed in a water bath at 37 °C (repeat this freeze/thaw cycle 3 times). The cell lysate was passed again through a 25 G needle (10 times).
  4. The lysates were then centrifuged at 400 x g for 8 min. The supernatant was recovered and the pellet discarded.
  5. Supernatant was transferred into a 15 ml tube, diluted with 2 volumes of CHAPS buffer 2x and then filtered using a 5 μm centrifugal filters at 2,000 x g during 10 min. The centrifugal filter was discarded and the filtrate kept.
  6. The filtrate was diluted with 1 volume of CHAPS buffer 2x and mixed by gentle pipetting up and down (this step is to lysate organelles such as mitochondria). Afterwards, the diluted lysate was centrifuged at 2,300 x g for 8 min and the pellet was recovered, discarding the supernatant.
  7. The pellet was re-suspended in 1 ml of CHAPS buffer 1x and centrifuged at 5,000 x g for 8 min, discarding the supernatants and keeping the pellet. Repeat this wash step twice.
  8. After the last centrifugation step, the pellet was re-suspended in 1 ml CHAPS buffer 1x and loaded carefully on the top of 200 µl of 40% Percoll (prepared previously in a 1.5 ml tube).


    Figure 1. Representative scheme of ASC and NLRP3 (p.D303N) particles purification protocol illustrating steps from 1 to 8

  9. Percoll was centrifuged at 16,000 x g for 10 min (turn off the break is not necessary). The interface layer containing the inflammasome particles was collected slowly and gently by pipetting and transferred into a new 1.5 ml tube and then washed with 1 ml of CHAPS buffer 1x by centrifugation using the same conditions (Figure 2).
  10. The supernatant was removed and the pellet was re-suspended in 100 µl of 1x CHAPS buffer (Figure 2).
  11. Finally, fluorescent particles were quantified in a fluorescence microscope using a Bürker chamber using a 1:10 dilution (this dilution depends on the concentration of recovered ASC or NLRP3 particles). After quantification the volume was adjusted until reach a density of 5 x 105 particles per µl.


    Figure 2. Representative scheme of Percoll step (steps 8-10)

Representative data

Table 1. Example of expected amounts of fluorescent particles recovered after purification protocol relative to 107 HEK293T cells expressing ASC-YFP or NLRP3 (p.D303N)-YFP. The cells were detached 48 h after transfection (cells expressing ASC-YFP) or until reach 90-100% of confluence per flask (cells stably expressing NLRP3(pD303N). The data shown are from experiments performed in different days.

Experiment
Asc-YFP (106 particles)
NLRP3 (p.D303N)-YFP (106 particles)
1
4.70
1.50
2
3.81
1.54
3
3.03
2.25
4
3.9
2.15
5
4.92
1.15
Average
4.07
1.72


Figure 3. Representative image of ASC and NLRP3 (p.D303N) fluorescent particles after purification protocol

Notes

  1. In step 2 cell pellet can be frozen at -80 °C (first in liquid nitrogen and then at -80 °C) or proceed with the speck purification protocol.
  2. Note that the supernatant recovered in step 4 after centrifugation is not clear.

Recipes

  1. 1x Percoll (to prepare 200 µl)
    120 µl 1x CHAPS
    80 µl Percoll-Plus
  2. Buffer A
    320 mM sucrose
    20 mM HEPES-KOH (pH 7.5)
    10 mM KCl
    1.5 mM MgCl2
    1 mM EDTA
    1 mM EGTA
  3. 2x CHAPS buffer
    40 mM HEPES-KOH (pH 7.5)
    10 mM MgCl2
    1 mM EGTA
    0.2 mM PMSF
    0.2 % CHAPS
    Note: 1x CHAPS buffer is prepared by diluting the CHAPS buffer 2x in distilled water.

Acknowledgments

This protocol has been adapted from the previously published paper: Baroja-Mazo et al. (2014). This work was supported by grants from PN I+D+I 2008-2011-Instituto Salud Carlos III-FEDER (PI13/00174) and European Research Council (ERC-2013-CoG 614578). The authors declare no conflict of interests.

References

  1. Baroja-Mazo, A., Martín-Sánchez, F., Gomez, A. I., Martínez, C. M., Amores-Iniesta, J., Compan, V., Barberà-Cremades, M., Yagüe, J., Ruiz-Ortiz, E. and Antón, J. (2014). The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15:738-48.
  2. Fernandes-Alnemri, T. and Alnemri, E. S. (2008). Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol 442: 251-270.
  3. Franklin, B. S., Bossaller, L., De Nardo, D., Ratter, J. M., Stutz, A., Engels, G., Brenker, C., Nordhoff, M., Mirandola, S. R. and Al-Amoudi, A. (2014). The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol 15(8): 727-737.
  4. Schroder, K. and Tschopp, J. (2010). The inflammasomes. Cell 140(6): 821-832.

简介

NLRP3炎症小体是负责炎症半胱天冬酶-1活化的多蛋白复合物,导致促炎细胞因子IL-1β和IL-18的加工和释放(Schroder和Tschopp,2010)。这种炎症小体由通过衔接蛋白ASC(凋亡相关的斑点样蛋白与半胱氨酸蛋白酶募集结构域)连接到caspase-1的传感蛋白NLRP3组成(Schroder和Tschopp,2010)。我们和其他人已经报道,在炎症小体激活时,NLRP3和ASC的功能性寡聚炎症小体颗粒从细胞中释放,充当危险信号,通过促进细胞外caspase-1的活化来扩增炎症(Baroja-Mazo等,
通过纯化ASC的重组颗粒或组成型激活的NLRP3突变体来研究寡聚ASC和NLRP3炎症小体颗粒的细胞外功能是可能的与相关的cryopyrin相关周期性综合征(CAPS,突变p.D303N),都标记有黄色荧光蛋白(YFP),并在HEK293细胞中表达。通过重组ASC或突变体NLRP3在HEK293细胞中的表达导致它们自发聚集成斑点(Baroja-Mazo等人,2014)的事实促进了纯化过程,并且方案最初从Fernandes-Alnemri和Alnemri(2008年)。

材料和试剂

  1. HEK293T细胞系(ATCC 号:CRL-11268)
  2. Dulbecco's Modified Eagle's medium F12(DMEM-F12)(Biowest,目录号:L0090)
  3. 10%(v/v)胎牛血清(Lonza,目录号:DE14-801F)
  4. 200mM L-谷氨酰胺(Lonza,目录号:17-605E)
  5. 青霉素和链霉素(Lonza,目录号:17-603E)
  6. 液氮
  7. 1x DPBS(Life Technologies,Gibco ,目录号:14190-094)
  8. 1x Percoll(见配方)
  9. 缓冲区A(参见配方)
  10. 2x CHAPS(请参阅配方)
  11. 1x CHAPS(请参阅配方)

设备

  1. 具有快速释放螺帽75通气的组织培养瓶(SARSTEDT AG,目录号:83.1813.002)
  2. 具有盖子,疏水性,无菌(SARSTEDT AG,目录号:83.1839)的组织培养板
  3. 37℃,5%CO 2细胞培养箱中培养
  4. 用于15ml Falcon管(Sigma-Aldrich,目录号:3K30)的带有转子的台式冷却离心机
  5. 带有用于2ml管(HERMLE,目录号:Z216MK)的转子的台式冷却微量离心机
  6. 37℃水浴
  7. 注射器(1ml)(Nipro注射器,目录号:SY31SCTU EC)
  8. 20 G和25 G针(Becton,Dickinson and Company,目录号:300600)
  9. 组织培养II级层流罩(Telstar Bio II)
  10. 带落射荧光的倒置显微镜(Nikon Eclipse Ti)
  11. 伯克计数室
  12. Ultrafree-CL低结合Durapore PVDF膜(5μm)(EMD Millipore,目录号:UFC40S25)

程序

除非另有说明,否则在冰上进行所有步骤,并在4°C下进行所有离心 开始从保持在补充有10%FCS,2mM Glutamax的DMEM:F12(1:1)中的瞬时表达ASC-YFP或稳定表达NLRP3(p.D303N)-YFP的10 7 HEK293的纯化, 1%青霉素 - 链霉素。

  1. 用温热的PBS洗涤细胞,并用6ml冷PBS通过上下吹吸瓶底部分离它们。 将细胞悬浮液转移到15ml管中(从1到8的步骤的代表性方案显示在图1中)。
  2. 通过在2,000xg下离心3分钟使细胞沉淀。 弃去上清液并将沉淀重悬在600μl冷的Buffer-A中,并转移到1.5ml管中
  3. 缓冲液A中的细胞在冰上通过具有20G针的1ml注射器(10次),然后通过25G针(20次)裂解。之后,将裂解物在液氮(在液氮中保持约30-40秒,或直到裂解物冷冻)中冷冻,随后在37℃的水浴中解冻(重复该冻/融循环3次)。细胞裂解物再次通过25G针(10次)。
  4. 然后将裂解物在400×g离心8分钟。回收上清液,弃去沉淀。
  5. 将上清液转移到15ml管中,用2体积的CHAPS缓冲液2x稀释,然后使用5μm离心过滤器在2,000xg下过滤10分钟。弃去离心过滤器,保留滤液
  6. 将滤液用1体积的CHAPS缓冲液2x稀释并通过温和地上下吹打混合(该步骤是裂解细胞器,例如线粒体)。然后,将稀释的裂解物在2,300×g离心8分钟,回收沉淀,弃去上清液。
  7. 将沉淀重悬于1ml CHAPS缓冲液1x中,并以5000×g离心8分钟,弃去上清液并保留沉淀。重复此洗涤步骤两次。
  8. 在最后一次离心步骤后,将沉淀物重新悬浮于1ml CHAPS缓冲液1x中,并小心地装载在200μl40%Percoll(预先制备在1.5ml管中)的顶部。


    图1. ASC和NLRP3(p.D303N)颗粒的纯化方案的代表性方案,说明从1到8的步骤

  9. 将Percoll在16,000×g离心10分钟(关闭断裂不是必需的)。通过吸移缓慢并轻轻地收集含有炎性体颗粒的界面层并转移到新的1.5ml管中,然后使用相同条件通过离心用1ml CHAPS缓冲液1x洗涤(图2)。
  10. 除去上清液,将沉淀重新悬浮于100μl1×CHAPS缓冲液中(图2)
  11. 最后,使用Bürker室使用1:10稀释液(该稀释度取决于回收的ASC或NLRP3颗粒的浓度)在荧光显微镜中定量荧光颗粒。定量后,调节体积直到达到每μl5×10 5个颗粒的密度。


    图2. Percoll步骤(步骤8-10)的代表方案

代表数据

表1.在纯化方案后回收的荧光颗粒相对于表达ASC-YFP或NLRP3(p.D303N)-YFP的10 7个HEK293T细胞的预期量的实施例。 在转染后48小时(表达ASC-YFP的细胞)或直到达到每个烧瓶(稳定表达NLRP3的细胞(pD303N)的90-100%汇合)分离细胞。所示数据来自在不同天进行的实验。

实验
Asc-YFP(10 6 粒子)
NLRP3(p.D303N)-YFP(10 6 粒子)
1
4.70
1.50
2
3.81
1.54
3
3.03
2.25
4
3.9
2.15
5
4.92
1.14
平均
4.07
1.72


图3.纯化方案后ASC和NLRP3(p.D303N)荧光颗粒的代表性图像

笔记

  1. 在步骤2中,可将细胞沉淀物在-80℃下冷冻(首先在液氮中然后在-80℃下)或用斑点纯化方案进行。
  2. 注意,离心后步骤4中回收的上清液不清楚

食谱

  1. 1x Percoll(制备200μl)
    120μl1x CHAPS
    80μlPercoll-Plus
  2. 缓冲区A
    320mM蔗糖 20mM HEPES-KOH(pH7.5) 10 mM KCl
    1.5mM MgCl 2·h/v 1mM EDTA
    1 mM EGTA
  3. 2x CHAPS缓冲区
    40mM HEPES-KOH(pH7.5) 10mM MgCl 2/
    1 mM EGTA
    0.2 mM PMSF
    0.2%CHAPS
    注意:1x CHAPS缓冲液通过用蒸馏水稀释CHAPS缓冲液2x来制备。

确认

该协议已经从先前发表的文章改编而来:Baroja-Mazo等人 (2014)。 这项工作得到了来自PN I + D + I 2008-2011-Salud Carlos III-FEDER研究所(PI13/00174)和欧洲研究理事会(ERC-2013-CoG 614578)的资助。 作者声明没有利益冲突。

参考文献

  1. Baroja-Mazo,A.,Martín-Sánchez,F.,Gomez,AI,Martínez,CM,Amores-Iniesta,J.,Compan,V.,Barberà-Cremades,M.,Yagüe,J.,Ruiz-Ortiz, E.和Antón,J。(2014)。 NLRP3炎症小体被释放为颗粒危险信号,放大 炎症反应。 Nat Immunol 15:738-48
  2. Fernandes-Alnemri,T。和Alnemri,E.S.(2008)。 ASC染色体的活性的装配,纯化和测定。 Methods Enzymol 442:251-270。
  3. Franklin,BS,Bossaller,L.,De Nardo,D.,Ratter,JM,Stutz,A.,Engels,G.,Brenker,C.,Nordhoff,M.,Mirandola,SR和Al-Amoudi, 2014)。 衔接子ASC具有增殖炎症的细胞外和"朊病毒"活性 。 Nat Immunol 15(8):727-737
  4. Schroder,K。和Tschopp,J。(2010)。 炎症小体。 140(6):821 -832。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martín-Sánchez, F., Gómez, A. I. and Pelegrín, P. (2015). Isolation of Particles of Recombinant ASC and NLRP3. Bio-protocol 5(10): e1480. DOI: 10.21769/BioProtoc.1480.
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