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Measurement of TACE Activity in Extracts from Cultured Cells
培养细胞提取物的肿瘤坏死因子α转化酶(TACE)活性测定   

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Abstract

In cigarette smoke–induced and inflammation-associated lung cancer development, cigarette smoke extract (CSE) activates tumor necrosis factor-alpha (TNF-α) secretion from macrophages. TNF-α converting enzyme (TACE), also known as α-Secretase or ADAM17 (A Disintegrin and Metalloprotease), is a member of the ADAM family of metalloproteases. TACE mediated ectodomain shedding leads to the conversion of the inactive TNF-α precursor into the active mature pro-inflammatory cytokine. The SensoLyte 520 TACE (α-Secretase) Activity Assay Kit was used to detect TACE activity in CSE-activated macrophages. This assay is reliable, reproducible and easy to carry out in 96 well plate format.

Keywords: TACE(TACE), TNF-alpha(肿瘤坏死因子), Cell culture(细胞培养), Extract(提取物), Secretion(分泌)

Materials and Reagents

  1. Cultured cells such as the macrophage line THP1
  2. SensoLyte 520 TACE (α-Secretase) Activity Assay Kit *Fluorimetric* (AnaSpec, catalog number: 72085 )
    Components used from the kit:
    1. Component A QXL™ 520/5-FAM (TACE substrate, Ex/Em = 490 nm/520 nm)
    2. Component B 5-FAM, fluorescence reference standard (Ex/Em = 490 nm/520 nm)
    3. Component C assay buffer
  3. Triton-X 100 (Sigma-Aldrich, catalog number: T8532 )
  4. Phosphate buffered saline (PBS) (see Recipes)

Equipment

  1. 96 well black microplate (for fluorescence analysis) (Greiner Bio-One GmbH, catalog number: 655077 )
  2. Tube/vial unidirectional rotator (Glas-Col, catalog number: 099A RD4512 )
  3. Cell lifter (polyethylene, sterile) (Corning Incorporated Costar, catalog number: 3008 )
  4. 1.5 ml microcentrifuge tubes (polypropylene) (USA Scientific, catalog number: 1615-5599 )
  5. Microplate fluorometer with detection range (excitation 490 nm and emission 520 nm) (settings recommended by SensoLyte 520 TACE Activity Assay Kit) (Thermo Fisher Scientific, Fluoroskan AscentTM) (used for TACE activity assay)

Procedure

Following Protocol B from the manufacturer’s manual and described the details below:

  1. Prepare cell lysates
    1. Amount of adherent cells needed for this assay are approximately 3 x 105 or 70 - 80% confluent cells per experimental sample in one well of 12 well plate.
    2. Add 0.1% (v/v) Triton-X 100 to Component C assay buffer.
    3. After induction of TACE activation with treatment of choice, remove media; wash cells with PBS; lift cells, collect into 1.5 ml microcentrifuge tube, and lyse with 200 µl Component C assay buffer. (The amount of assay buffer needs to be determined empirically for the experimental samples used.)
    4. Incubate the cell suspension at 4 °C for 10 min on unidirectional rotator.
    5. Centrifuge the lysed cells at 2,500 x g at 4 °C.
    6. Collect supernatant containing protein sample into a new 1.5 ml microcentrifuge tube.
  2. Prepare working solutions
    Note: All kit components should be thawed to room temperature before starting the experiment. Protect light sensitive materials from exposure to ambient light.
    For TACE substrate solution, dilute TACE substrate (Component A) 1:100 in assay buffer (Component C).
    Note: For each experiment, prepare fresh substrate solution.
  3. Set up TACE enzymatic reaction
    1. Set up at least triplicate enzymatic reactions.
    2. Add 50 µl of protein sample (supernatant collected in step 1f) into black 96 well plate.
    3. Set up a 50 µl blank control containing assay buffer (Component C) only. The blank control well fluorescence reading accounts for the background fluorescence contributed by the assay buffer. In this case, the blank control functions as the reagent blank consisting only of the assay buffer used to lyse the cells; in general, the absorbance reading for this solution is typically subtracted from each sample absorbance reading in order to only consider the substance you are trying to analyze or measure.
    4. In the dark, set up a 50 µl positive control with the 5-FAM fluorescence reference standard: Dilute 1 mM 5-FAM (Component B) to 4 μM in assay buffer (Component C). Do 2-fold serial dilutions to get concentrations of 2, 1, 0.5, 0.25, 0.12, and 0.06 μM, include an assay buffer blank. Add 50 μl per well of these serially diluted 5-FAM reference solutions. Protect tubes and plate from ambient light.
    5. On lab bench, pre-incubate the plate for 10 min at room temperature protected from ambient light.
  4. Measure TACE activity
    Use TACE substrate solution equilibrated to room temperature. Add 50 µl of TACE substrate solution into each well (each well now contains a total volume of 100 µl). Mix reagents in plate by gently shaking by hand for 30 sec.
    Immediately quantify TACE enzymatic activity by measuring fluorescence intensity at Ex/Em=485 nm/538 nm.
  5. Detect protein concentration
    Use a protein quantification method, such as the Bradford assay, to measure total protein concentration in each experimental sample from the cell lysates prepared in steps 1c-f (He, 2011).
  6. Data analysis
    The blank control well fluorescence reading accounts for the background fluorescence contributed by the assay buffer. Subtract the background fluorescence reading of the blank from each sample in order to only consider the TACE activity measured. Normalize the relative fluorescence units (RFU) of each experimental sample to its total protein concentration. Plot the data as TACE activity RFU/ µg protein as shown in Figure 1.


    Figure 1. An example of plot of TACE activity (RFU/μg protein)

Notes

  1. Ex/Em=485 nm/538 nm was used to detect fluorescence in cell lysates. Settings were modified from those recommended by SensoLyte 520 TACE Activity Assay Kit (excitation 490 nm and emission 520 nm) because the Fluoroskan AscentTM fluorescence plate reader used in this experiment is limited to this pair of excitation/emission settings.
  2. Measuring TACE activity may be repeated several times from 0 to 15 min to capture an optimal reading. Kinetic reading of TACE activity should be empirically determined for your experimental system.

Recipes

  1. Phosphate buffered saline [PBS, 0.9% (w/v) sodium chloride in 10 mM phosphate buffer]
    NaCl
    8.00 g
    KCl
    0.20 g
    Na2HPO4
    1.44 g
    KH2PO4
    0.24 g
    Deionized water 800 ml
    Adjust pH to 7.4 with HCl or NaOH
    Deionized water to 1 L

Acknowledgments

This protocol was adapted from a previously published paper Xu et al. (2014). This study was partly supported by grants from NIEHS/NIH (R01ES017328), NCI/NIH (R01CA142649), and the Office of Science (BER), U.S. Department of Energy (DE-FG02-09ER64783).

References

  1. He, F. (2011). Bradford Protein Assay. Bio-protocol 1(6): e45.
  2. Xu, X., Padilla, M. T., Li, B., Wells, A., Kato, K., Tellez, C., Belinsky, S. A., Kim, K. C. and Lin, Y. (2014). MUC1 in macrophage: contributions to cigarette smoke-induced lung cancer. Cancer Res 74(2): 460-470.

简介

在香烟烟雾诱导和炎症相关的肺癌发展中,香烟烟雾提取物(CSE)激活巨噬细胞的肿瘤坏死因子-α(TNF-α)分泌。 TNF-α转化酶(TACE),也称为α-分泌酶或ADAM17(解聚素和金属蛋白酶),是金属蛋白酶的ADAM家族的成员。 TACE介导的胞外结构域脱落导致无活性的TNF-α前体转化为活性成熟促炎细胞因子。 SensoLyte 520 TACE(α-分泌酶)活性测定试剂盒用于检测CSE活化的巨噬细胞中的TACE活性。 该测定法是可靠的,可重复的并且容易在96孔板形式中进行。

关键字:TACE, 肿瘤坏死因子, 细胞培养, 提取物, 分泌

材料和试剂

  1. 培养的细胞如巨噬细胞系THP1
  2. SensoLyte 520 TACE(α-分泌酶)活性测定试剂盒*荧光*(AnaSpec,目录号:72085)
    套件中使用的组件:
    1. 组分A QXL TM 520/5-FAM(TACE底物,Ex/Em = 490nm/520nm)
    2. 组分B 5-FAM,荧光参比标准品(Ex/Em = 490nm/520nm)
    3. 组分C测定缓冲液
  3. Triton-X 100(Sigma-Aldrich,目录号:T8532)
  4. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. 96孔黑色微孔板(用于荧光分析)(Greiner Bio-One GmbH,目录号:655077)
  2. 管/小瓶单向旋转器(Glas-Col,目录号:099A RD4512)
  3. 细胞提升器(聚乙烯,无菌)(Corning Incorporated Costar,目录号:3008)
  4. 1.5ml微量离心管(聚丙烯)(USA Scientific,目录号:1615-5599)
  5. 具有检测范围(激发490nm和发射520nm)(由SensoLyte 520 TACE活性测定试剂盒推荐的设置)(Thermo Fisher Scientific,Fluoroskan Ascent TM)的微板荧光计(用于TACE活性测定)

程序

遵循制造商手册中的方案B,并详细描述如下:

  1. 准备细胞裂解液
    1. 该测定所需的贴壁细胞的量为12孔板的一个孔中每个实验样品的约3×10 5个或70〜80%汇合的细胞。
    2. 向组分C测定缓冲液中加入0.1%(v/v)Triton-X 100
    3. 用选择的治疗诱导TACE活化后,除去培养基; 用PBS洗涤细胞; 提起细胞,收集到1.5ml微量离心管中,并用200μl组分C测定缓冲液裂解。 (对于所用的实验样品,需要根据经验确定测定缓冲液的量。)
    4. 在4℃下在单向旋转器上孵育细胞悬浮液10分钟
    5. 在4℃下以2,500xg离心裂解的细胞。
    6. 将含有蛋白质样品的上清液收集到新的1.5ml微量离心管中
  2. 准备工作解决方案
    注意:所有试剂盒组件应在开始实验前解冻至室温。 保护光敏材料免受环境光照射。
    对于TACE底物溶液,在测定缓冲液(组分C)中稀释TACE底物(组分A)1:100 注意:对于每个实验,准备新的底物溶液。
  3. 建立TACE酶反应
    1. 设置至少三次酶反应。
    2. 加入50μl蛋白质样品(步骤1f收集的上清液)到黑色96孔板中
    3. 设置50微升空白对照含有测定缓冲液(组分C)。空白对照孔荧光读数考虑了由测定缓冲液贡献的背景荧光。在这种情况下,空白对照用作仅由用于裂解细胞的测定缓冲液组成的试剂空白;一般来说,通常从每个样品的吸光度读数中减去该溶液的吸光度读数,以便仅考虑您试图分析或测量的物质。
    4. 在黑暗中,用5-FAM荧光参考标准品设置50μl阳性对照:在测定缓冲液(组分C)中稀释1mM 5-FAM(组分B)至4μM。进行2倍连续稀释以获得2,1,0.5,0.25,0.12和0.06μM的浓度,包括测定缓冲液空白。每孔加入50μl这些系列稀释的5-FAM参比溶液。保护管和板免受环境光照。
    5. 在实验室工作台上,将板在室温下预温育10分钟,避免环境光照射
  4. 测量TACE活动
    使用平衡至室温的TACE底物溶液。添加50微升的TACE底物溶液到每个孔(每个井现在包含总体积100微升)。通过用手轻轻摇动30秒来混合板中的试剂。
    通过在Ex/Em = 485nm/538nm处测量荧光强度立即定量TACE酶活性。
  5. 检测蛋白质浓度
    使用蛋白质定量方法,例如Bradford测定,测量在步骤1c-f(He,2011)中制备的细胞裂解物的每个实验样品中的总蛋白质浓度。
  6. 数据分析
    空白对照孔荧光读数考虑了由测定缓冲液贡献的背景荧光。 减去每个样品的空白的背景荧光读数,以便仅考虑测量的TACE活性。 将每个实验样品的相对荧光单位(RFU)标准化为其总蛋白浓度。 将数据绘制为TACE活性RFU /μg蛋白质,如图1所示

    图1. TACE活性(RFU /μg蛋白)的图示例

笔记

  1. Ex/Em = 485nm/538nm用于检测细胞裂解物中的荧光。 设置从SensoLyte 520 TACE活性测定试剂盒(激发490nm和发射520nm)推荐的那些修改,因为在该实验中使用的Fluoroskan Ascent TM荧光读板仪限于这对激发/发射 设置。
  2. 测量TACE活性可以从0至15分钟重复几次以捕获最佳读数。 TACE活性的动力学读数应该根据您的实验系统经验确定。

食谱

  1. 磷酸盐缓冲盐水[PBS,0.9%(w/v)氯化钠的10mM磷酸盐缓冲液]
    NaCl
    8.00克
    KCl
    0.20克
    Na HPO 4
    1.44克
    KH 2 PO 4
    0.24克
    去离子水800 ml
    用HCl或NaOH调节pH至7.4 去离子水至1升

致谢

该协议改编自以前发表的论文Xu et al。(2014)。 本研究部分得到NIEHS/NIH(R01ES017328),NCI/NIH(R01CA142649)和美国能源部(DE-FG02-09ER64783)科学办公室(BER)的资助。  

参考文献

  1. 他,F.(2011)。 Bradford Protein Assay 生物协议 1(6):e45。
  2. Xu,X.,Padilla,M.T.,Li,B.,Wells,A.,Kato,K.,Tellez,C.,Belinsky,S.A.,Kim,K.C.and Lin, 巨噬细胞中的MUC1:对香烟烟雾诱导的肺癌的贡献 癌症 Res 74(2):460-470。
  • English
  • 中文翻译
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xu, X., Padilla, M. T. and Lin, Y. (2014). Measurement of TACE Activity in Extracts from Cultured Cells. Bio-protocol 4(20): e1264. DOI: 10.21769/BioProtoc.1264.
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