欢迎您, 登录 | 注册

首页 | English

X
加载中

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).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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

免疫学 > 宿主防御 > 综合
作者: Fátima Martín-Sánchez
Fátima Martín-SánchezAffiliation: Inflammation and Experimental Surgery Unit, Clinical University Hospital "Virgen de la Arrixaca", Murcia, Spain
For correspondence: fatima.martin@ffis.es
Bio-protocol author page: a2233
Ana I. Gómez
Ana I. GómezAffiliation: Inflammation and Experimental Surgery Unit, Clinical University Hospital "Virgen de la Arrixaca", Murcia, Spain
Bio-protocol author page: a2234
 and Pablo Pelegrín
Pablo PelegrínAffiliation 1: Inflammation and Experimental Surgery Unit, Clinical University Hospital "Virgen de la Arrixaca", Murcia, Spain
Affiliation 2: CIBERehd, Murcia’s BioHealth Research Institute IMIB-Arrixaca, Clinical University Hospital "Virgen de la Arrixaca", Murcia, Spain
For correspondence: pablo.pelegrin@ffis.es
Bio-protocol author page: a326
Vol 5, Iss 10, 5/20/2015, 1751 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1480

[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.

Acknowledgement

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.


How to cite this protocol: 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; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook