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Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay
使用分泌型胚胎碱性磷酸酶NF-κB报告基因实验筛选新型内源性炎症刺激物质

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Abstract

An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.

Keywords: Innate immunity(先天免疫), TLR4(TLR4), Tenascin-C(腱生蛋白-C), LPS(LPS), THP1-BlueTM cells(THP1-BlueTM细胞), NF-κB reporter(NF-κB报告基因)

Background

The immune system has evolved to recognize not only pathogenic stimuli such as bacterial components and viral nucleic acids, but also endogenous danger signals including proteins secreted from necrotic cells or expressed upon tissue damage. Both types of stimuli are sensed by pattern recognition receptors, initiating signalling cascades that trigger inflammatory responses. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor essential for the activation of the immune response to infection and tissue damage. NF-κB has an important role in the expression of a wide range of inflammatory mediators following pattern recognition receptor activation, including cytokines (such as tumour necrosis factor α [TNFα], interleukin-6 and interleukin-1), chemokines (e.g., interleukin-8 or CXCL1), proteases, growth factors and MHC-related molecules, among others. To assess the activation of this pathway downstream of TLR4, we used the commercially available cell line THP1-BlueTM NF-κB (Invivogen). These cells are stably transfected with a construct containing a secreted embryonic alkaline phosphatase (SEAP) gene induced by the NF-κB transcription factor. The construct contains an interferon-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site, which drives the expression of the reporter gene. After cell stimulation with pathogenic or endogenous stimuli, NF-κB activation leads to the secretion of SEAP, which is then quantified using the colorimetric reagent QUANTI-BlueTM. This is a quick and reliable method to assess activation of NF-κB downstream of toll-like receptors, as the amount of SEAP in the media correlates with the triggering of this signalling pathway. However, this is an engineered cell line that does not express a full complement of inflammatory effector molecules (see Note 5), and so whilst useful in screening for NF-κB activation, data should always be confirmed in additional experimental systems, for example in primary macrophages, or in vivo.

Materials and Reagents

  1. Pipette tips  
    20 µl (StarLabs, TipOne®, catalog number: S1110-3800 )
    200 µl (StarLabs, TipOne®, catalog number: S1111-0806 )
    1,000 µl (StarLabs, TipOne®, catalog number: S1111-6801 )
  2. Corning 15 ml PP centrifuge sterile tubes (Corning, catalog number: 430791 )
  3. Cell culture flask 75 cm2 (VWR, catalog number: 734-0012 )
  4. 96 well plate flat-bottom (VWR, catalog number: 734-0023 )
  5. THP1-BlueTM NF-κB cells (InvivoGen, catalog number: thp-nfkb )
  6. Positive control: LPS from E. coli, Serotype EH100 (Ra) (TLRgradeTM) (Enzo life sciences, catalog number: ALX-581-010-L002 )
  7. Test inflammatory stimulus: for example recombinant tenascin-C protein or the fibrinogen like globe domain (FBG) of tenascin-C protein (synthesized as described in Midwood et al., 2009)
  8. Roswell Park Memorial Institute (RPMI) 1640 with L-glutamine (Lonza, catalog number: BE12-702F )
  9. Fetal bovine serum (FBS), Qualified, heat inactivated (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  10. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  11. NormocinTM (InvivoGen, catalog number: ant-nr-1 )
  12. Blasticidin HCl (Thermo Fisher Scientific, GibcoTM, catalog number: R21001 )
  13. QUANTI-BlueTM (InvivoGen, catalog number: rep-qb1 )
  14. ThP1 media without blasticidin (see Recipes)
  15. ThP1 media with blasticidin (see Recipes)
  16. QUANTI-BlueTM solution (see Recipes)

Equipment

  1. P20, P200 and P1000 pipettes
  2. 37 °C water bath
  3. Hemocytometer
  4. CO2 incubator
  5. FluoStar Omega plate reader
  6. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM MAgefugeTM 16 )
  7. Rotor (Thermo Fisher Scientific, Thermo ScientificTM, model: TX-400 4 x 400 mL Swinging Bucket Rotor , catalog number: 75003629)

Software

  1. Graph Pad Prism

Procedure

  1. Cell culture conditions
    1. Thaw a vial containing 3-7 x 106 THP1-BlueTM NF-κB cells in a 37 °C water bath.
    2. Transfer cells to a 15 ml sterile tube containing 10 ml of pre-warmed ThP1 NF-κB medium without blasticidin (see Recipes). Centrifuge tube at 320 x g for 5 min.
    3. Remove supernatant and resuspend cells in 10 ml of ThP1 NF-κB medium without blasticidin. Count the cells using a hemocytometer.
    4. Transfers 1 x 106 cells in 10 ml of ThP1 NF-κB medium without blasticidin to a 75 cm2 cell culture flask and place the culture at 37 °C in 5% CO2.
    5. The cells grow in suspension. To passage the cells, transfer cells to a sterile 15 ml tube and measure cell concentration using a hemocytometer. Centrifuge tube at 320 x g for 5 min at room temperature, remove cell supernatant and resuspend cells in fresh media. Passage the cells every 3 days and do not let the cell concentration in the flask exceed 2 x 106 cells/ml.
    6. For the first two passages, cells should be cultured in medium without blasticidin to allow recovery from freezing. After the second passage, culture the cells in ThP1 NF-κB medium with blasticidin (see Recipes) to maintain selection pressure.

  2. Cell stimulation
    1. Transfer cells to a 15 ml sterile tube. Centrifuge cells at 320 x g for 5 min. Remove supernatant and resuspend cells in new ThP1 NF-κB medium with blasticidin.
    2. Plate 1 x 105 cells per well in a flat-bottom 96 well plate in a final volume of 100 µl.
    3. Immediately after plating, stimulate cells by adding LPS or recombinant protein (e.g., FBG) diluted in 100 µl of media in duplicate or triplicate wells. The final volume in each well is 200 µl.
    4. Incubate the cells for 24 h at 37 °C in 5% CO2 (see Note 3).
    5. To measure NF-κB-induced secreted alkaline phosphatase, collect cell supernatant.
    6. At this stage cells can be taken for assessment of viability for example by MTT assay.

  3. Measurement of NF-κB activation
    1. Warm QUANTI-BlueTM solution (see Recipes) at 37 °C for 10 min.
    2. Add 180 µl of QUANTI-BlueTM per well to a flat-bottom 96 well plate.
    3. Add 20 µl of THP1-BlueTM cell supernatant to each well.
    4. Incubate the plate at 37 °C for 2 h. The media will change color from pink to blue in the presence of secreted embryonic alkaline phosphatase (Figure 1).
    5. Determine SEAP levels by reading the optical density (OD) at 620 nm using a spectrophotometer plate reader.


      Figure 1. Detection of secreted embryonic alkaline phosphatase using QUANTI-BlueTM. Activation of THP1-BlueTM NF-κB cells with decreasing doses of LPS plus negative control (NC) performed in duplicates. In the presence of SEAP the QUANTI-BlueTM media changes color from pink to blue. The blue color intensity correlates with NF-κB activation at different doses.

Data analysis

For each stimulus, plot the mean of OD values ± standard error of the mean (SEM), in the case of multiple independent experiments (Figure 2), or the mean of OD values ± standard deviation (SD), in the case of single experiments. The data can also be represented as fold change difference compared to non-stimulated cells. Perform statistical analysis using Graph Pad Prism or other software.


Figure 2. NF-κB activation by LPS or the FBG domain of tenascin-C (FBG-C) in THP1-BlueTM NF-κB cells. Cells were stimulated with different doses of LPS or FBG-C or left unstimulated (-) and after 24 h NF-κB activation was measured using QUANTI-BlueTM. Data shown as mean ± SEM, N = 3 independent experiments. One-way ANOVA vs. non-stimulated with Dunnett’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis was performed using Graph Pad Prism.

Notes

  1. QUANTI-BlueTM signal can be detected between 15 min to 24 h after adding cell supernatant. Determine the optimal time point for measuring QUANTI-BlueTM signal to be able to compare between independent experiments.
  2. THP1-BlueTM NF-κB cells respond robustly to TLR1/2, TLR2/6, TLR4, TLR5, TLR8, NOD1 and NOD2 agonists. However, poor responses have been observed with TLR3, TLR7 and TLR9 ligands.
  3. Robust SEAP signal can be detected from 8 h after cell stimulation with TLR4 ligands (Figure 3). Nevertheless, it is recommended to titrate the stimulus of interest to determine the optimal dose and time point after stimulation to assess NF-κB activation.


    Figure 3. Time course of NF-κB activation in THP1-BlueTM NF-κB cells. Cells were stimulated with 1 ng/ml of LPS or 1 μM FBG-C and NF-κB activation was measured using QUANTI-BlueTM at different time points. Data shown as mean ± SEM, N = 3 independent experiments. One-way ANOVA vs. time 0 h with Dunnett’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001.

  4. THP1-BlueTM NF-κB cells should not be used after the 12th passage (Figure 4). According to the manufacturer (Invivogen), these cells undertake genotypic changes that result in reduced responsiveness over time under normal culture conditions. It is important to prepare frozen stocks at early passages.


    Figure 4. Measurement of NF-κB activation in THP1-BlueTM NF-κB cells at different passages. Cells from different passages were stimulated with 0.5 ng/ml of LPS or left unstimulated (-) and NF-κB activation was measured after 24 h using QUANTI-BlueTM. Data shown as mean ± SEM, N = 3. Paired t-test vs. non-stimulated, *P < 0.05, **P < 0.01, ***P < 0.001.

  5. THP1-BlueTM NF-κB cells cannot be used to assess cytokine protein expression. No cytokines can be reliably detected in the supernatant of these cells after activation with LPS (Figure 5).


    Figure 5. THP1-BlueTM NF-κB cells do not express cytokines. Cells were stimulated with different doses of LPS or left unstimulated (-) and NF-κB activation was measured after 24 h using QUANTI-BlueTM; or cytokine synthesis (IL-8, TNF and IL-6) was measured by ELISA. Data shown as mean ± SD, N = 2 independent experiments.

  6. When testing the inflammatory activity of recombinant proteins, the LPS content of preparations should be < 10 pg/ml. Additional controls that should be included are 1) incubation of the recombinant protein with polymyxin B, an antibiotic that inhibits pathogenic activation of TLR4, but which doesn’t affect the recombinant protein activity; and 2) boiling the samples, which doesn’t affect LPS activity, but denatures the protein, destroying its pro-inflammatory activity (Figure 6).


    Figure 6. Effect of polymyxin B (PMB) and boiling on LPS and FBG-C activity in THP1-BlueTM NF-κB cells. Cells were left unstimulated (-) or stimulated with LPS or FBG-C pre-incubated with PMB for 30 min or boiled for 15 min. NF-κB activation was measured using QUANTI-BlueTM. Data shown as mean ± SEM, N = 4 independent experiments. Paired t-test vs. non-treated, ***P < 0.001.

Recipes

  1. ThP1 media without blasticidin
    RPMI 1640
    10% FBS
    1% penicillin/streptomycin
    100 µg/ml NormocinTM
  2. ThP1 media with blasticidin
    RPMI 1640
    10% FBS
    1% penicillin/streptomycin
    10 µg/ml blasticidin
    100 µg/ml NormocinTM
  3. QUANTI-BlueTM solution
    One pouch of QUANTI-BlueTM
    100 ml of endotoxin-free water
    Pour the content of one pouch of QUANTI-BlueTM in a sterile flask with 100 ml of endotoxin-free water. Incubate QUANTI-BlueTM solution at 37 °C for 30 min and swirl gently every 10 min to dissolve the powder. QUANTI-BlueTM solution is stable for 2 weeks at 2-8 °C, or two month at -20 °C. Keep reconstituted QUANTI-BlueTM away from light

Acknowledgments

We thank Anna Marzeda for providing the image used in Figure 1. This work was supported by funding from Arthritis Research UK and the Kennedy Trust for Rheumatology Research. This protocol was first published in Piccinini et al. (2016) and also features in the following manuscript currently under review (Zuliani-Alvarez et al., 2017). The authors declare that they have no conflicts of interest or competing interests that impacted the design and implementation of this protocol.

References

  1. Midwood, K., Sacre, S., Piccinini, A. M., Inglis, J., Trebaul, A., Chan, E., Drexler, S., Sofat, N., Kashiwagi, M., Orend, G., Brennan, F., and Foxwell, B. (2009). Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nat Medi 15(7): 774-780.
  2. Piccinini, A. M., Zuliani-Alvarez, L., Lim, J. M. and Midwood, K. S. (2016). Distinct microenvironmental cues stimulate divergent TLR4-mediated signaling pathways in macrophages. Sci Signal 9(443): ra86.
  3. Zuliani-Alvarez, L. et al. (2017). Unravelling molecular determinants of danger: how tenascin-C is detected by toll-like receptor 4 (in review).

简介

免疫反应可以被致病性刺激以及内源性危险信号激活,引发模式识别受体的激活并引发导致炎症的信号级联。该方法使用THP1-Blue TM 细胞,其是含有能够检测NF-κB诱导的转录激活的胚胎碱性磷酸酶报告基因的人单核细胞系。通过两种不同的激动剂刺激toll样受体4(TLR4)后,通过评估NF-κB活化来验证该方案:从革兰氏阴性细菌的细胞壁衍生的脂多糖(LPS)和腱生蛋白C,细胞外基质蛋白其组织损伤引起其表达。然后我们使用该方案筛选潜在的新的内源性TLR4激动剂,但是该方法也可以用作快速,经济和可靠的方法来测定导致TLR依赖性NF-κB活化的其它炎性刺激的活性。

免疫系统已经发展成不仅识别病原性刺激物,例如细菌成分和病毒核酸,还包括内源性危险信号,包括从坏死细胞分泌的蛋白质或表达于组织损伤。通过模式识别受体感测到两种类型的刺激,启动触发炎症反应的信号级联。活化的B细胞(NF-κB)的核因子κ-轻链增强子是激活对感染和组织损伤的免疫应答所必需的转录因子。 NF-κB在模式识别受体激活后,在广泛的炎症介质的表达中具有重要作用,包括细胞因子(如肿瘤坏死因子α[TNFα],白细胞介素-6和白细胞介素-1),趋化因子例如白细胞介素-8或CXCL1),蛋白酶,生长因子和MHC相关分子等。为了评估在TLR4下游的该途径的活化,我们使用市售的细胞系THP1-Blue NF-κB(Invivogen)。这些细胞用含有由NF-κB转录因子诱导的分泌的胚胎碱性磷酸酶(SEAP)基因的构建体稳定转染。该构建体含有融合到5拷贝的NF-κB共有转录应答元件的干扰素-β最小启动子和c-Rel结合位点的3个拷贝,其驱动报告基因的表达。在用致病或内源性刺激进行细胞刺激后,NF-κB活化导致SEAP的分泌,然后使用比色试剂QUANTI-Blue 定量。这是一种快速可靠的方法来评估细胞周期受体下游的NF-κB的活化,因为培养基中SEAP的量与该信号通路的触发相关。然而,这是一种工程细胞系,其不表达炎性效应分子的完整补体(参见附注5),因此虽然可用于筛选NF-κB活化,但数据应始终在额外的实验系统中得到证实,例如原代巨噬细胞或体内。

关键字:先天免疫, TLR4, 腱生蛋白-C, LPS, THP1-BlueTM细胞, NF-κB报告基因

材料和试剂

  1. 移液器提示
    20μl(StarLabs,TipOne ®,目录号:S1110-3800)
    200μl(StarLabs,TipOne ®,目录号:S1111-0806)
    1000μl(StarLabs,TipOne ®,目录号:S1111-6801)
  2. Corning 15 ml PP离心机无菌管(Corning,目录号:430791)
  3. 细胞培养瓶75厘米2(VWR,目录号:734-0012)
  4. 96孔板平底(VWR,目录号:734-0023)
  5. THP1-BlueTM NF-κB细胞(InvivoGen,目录号:thp-nfkb)
  6. 阳性对照:来自E的LPS。大肠杆菌,血清型EH100(Ra)(TLRgrade TM)(Enzo生命科学,目录号:ALX-581-010-L002)
  7. 测试炎症刺激:例如重组腱生蛋白-C蛋白或腱生蛋白-C蛋白的纤维蛋白原样球状结构域(FBG)(如Midwood等人,2009中所述合成)
  8. 罗斯韦尔公园纪念研究所(RPMI)1640与L-谷氨酰胺(Lonza,目录号:BE12-702F)
  9. 胎牛血清(FBS),合格的,热灭活的(Thermo Fisher Scientific,Gibco TM,目录号:10500064)
  10. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  11. Normocin TM (InvivoGen,目录号:ant-nr-1)
  12. 盐酸杀菌素(Thermo Fisher Scientific,Gibco TM,目录号:R21001)
  13. QUANTI-Blue TM (InvivoGen,目录号:rep-qb1)
  14. 不含杀稻瘟素的ThP1培养基(见食谱)
  15. ThP1介质与杀稻瘟素(见食谱)
  16. QUANTI-Blue TM 解决方案(请参阅食谱)

设备

  1. P20,P200和P1000移液器
  2. 37°C水浴
  3. 血细胞计数器
  4. CO 2 孵化器
  5. FluoStar欧米茄读卡器
  6. 离心机(Thermo Fisher Scientific,型号:Heraeus TM MAgefuge TM 16)
  7. 转子(Thermo Fisher Scientific,Thermo Scientific TM,型号:TX-400 4 x 400 mL摇摆转筒,目录号:75003629)

软件

  1. 图垫棱镜

程序

  1. 细胞培养条件
    1. 在37℃水浴中解冻含有3-7×10 6个THP1-Blue TM NF-κB细胞的小瓶。
    2. 将细胞转移到含有10ml预热的ThP1 NF-κB培养基的15ml无菌管中,不含杀稻瘟素(见食谱)。离心管320×g,持续5分钟。
    3. 除去上清液并将细胞重悬于10ml无杀稻瘟素的ThP1 NF-κB培养基中。使用血细胞计数器计数细胞。
    4. 将1×10 6个细胞在不含杀稻瘟素的10毫升ThP1 NF-κB培养基中转移至75厘米2细胞培养瓶中,并将培养物在37℃下放置5% CO 2
    5. 细胞生长悬浮。为了通过细胞,将细胞转移到无菌的15ml管中,并使用血细胞计数器测量细胞浓度。在室温下将320g离心管放置5分钟,除去细胞上清液并将细胞重新悬浮于新鲜培养基中。每3天通过细胞,不要让烧瓶中的细胞浓度超过2×10 6细胞/ml。
    6. 对于前两代,细胞应在不含杀稻瘟菌素的培养基中培养,以使其从冷冻中恢复。第二次传代后,用杀稻瘟素培养ThP1 NF-κB培养基中的细胞(参见食谱)以维持选择压力。

  2. 细胞刺激
    1. 将细胞转移到15 ml无菌管中。以320g离心细胞5分钟。去除上清液并将细胞重新悬浮于新的具有杀稻瘟素的ThP1 NF-κB培养基中
    2. 在平底96孔板中以100μl的最终体积每孔1×10 5个细胞。
    3. 电镀后,立即通过加入稀释在100μl一式两份或一式三份孔中的培养基中的LPS或重组蛋白(例如,FBG)来刺激细胞。每个孔的最终体积为200μl。
    4. 在37℃下在5%CO 2中孵育细胞24小时(见注3)。
    5. 测量NF-κB诱导的分泌性碱性磷酸酶,收集细胞上清液
    6. 在这个阶段,细胞可以用于评估生存力,例如通过MTT测定
  3. NF-κB活化的测定


























    1. 每孔加入180μlQUANTI-Blue TM至平底96孔板。
    2. 向每个孔中加入20μl的THP1-Blue TM细胞上清液。
    3. 在37℃孵育2小时。在分泌的胚胎碱性磷酸酶的存在下,培养基将从粉红色变为蓝色(图1)
    4. 通过使用分光光度计读板器读取620nm处的光密度(OD)来确定SEAP水平

      图1.使用QUANTI-Blue 检测分泌的胚胎碱性磷酸酶。以降低剂量的活性激活THP1-Blue NF-κB细胞LPS加阴性对照(NC)重复进行。在SEAP的存在下,QUANTI-Blue TM 媒体将颜色从粉红色变为蓝色。蓝色强度与不同剂量的NF-κB活化相关

数据分析

对于每个刺激,绘制OD值的平均值±平均值的标准误差(SEM),在多个独立实验(图2)的情况下,或OD值的平均值±标准偏差(SD),在单个情况下实验。数据也可以表示为与非刺激细胞相比的折叠变化差异。使用Graph Pad Prism或其他软件进行统计分析。


图2.在THP1-Blue NF-κB细胞中,通过LPS或腱生蛋白-C(FBG-C)的FBG结构域的NF-κB活化。
细胞用使用QUANTI-Blue 测量不同剂量的LPS或FBG-C或未被刺激的( - )和24小时NF-κB活化后。数据显示为平均值±SEM,N = 3次独立实验。单因素方差分析与Dunnett的事后检验无刺激相关, 0.05,** P 0.01, *** P 0.001。使用Graph Pad Prism进行统计分析。

笔记

  1. QUANTI-Blue TM 信号可以在加入细胞上清后15分钟至24小时检测。确定测量QUANTI-Blue TM 信号的最佳时间点,以便能够在独立实验之间进行比较。
  2. TLP1-Blue TM NF-κB细胞对TLR1/2,TLR2/6,TLR4,TLR5,TLR8,NOD1和NOD2激动剂的稳健反应。然而,TLR3,TLR7和TLR9配体的反应差。
  3. 可以在TLR4配体细胞刺激后8 h检测到健壮的SEAP信号(图3)。然而,建议滴定感兴趣的刺激物,以确定刺激后评估NF-κB活化的最佳剂量和时间点。


    图3:THP1-Blue NF-κB细胞中NF-κB活化的时间过程细胞用1ng/ml LPS或1μMFBG-C并且在不同时间点使用QUANTI-Blue TM测量NF-κB活化。数据显示为平均值±SEM,N = 3次独立实验。单因素方差分析与时间0小时,Dunnett的事后检验,* 0.05,** P < 0.01,*** 0.001。

    致谢

    我们感谢Anna Marzeda提供图1中使用的图像。这项工作得到了英国关节炎研究和肯尼迪风湿病研究信托基金的支持。该协议首次在Piccinini等人发布。 (2016),并且还在以下正在审查中的手稿(Zuliani-Alvarez等人,2017年)中具有特征。作者宣称它们没有影响本协议的设计和实施的利益冲突或竞争利益。

    参考文献

    1. Midwood,K.,Sacre,S.,Piccinini,AM,Inglis,J.,Trebaul,A.,Chan,E.,Drexler,S.,Sofat,N.,Kashiwagi,M.,Orend,G.,Brennan ,F.和Foxwell,B.(2009)。腱生蛋白-C是Toll样受体4的内源性激活剂,其对于维持关节炎关节疾病中的炎症是必需的。 15(7):774-780。 >
    2. Piccinini,AM,Zuliani-Alvarez,L.,Lim,JM和Midwood,KS(2016)。  独特的微环境提示刺激巨噬细胞中不同的TLR4介导的信号通路。 9(443):ra86。
    3. Zuliani-Alvarez,L.等人。 (2017)。揭示危险的分子决定因素:如何通过收费样受体4检测腱生蛋白C(综述)。
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引用:Zuliani-Alvarez, L., Piccinini, A. M. and Midwood, K. S. (2017). Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay. Bio-protocol 7(7): e2220. DOI: 10.21769/BioProtoc.2220.
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