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Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader
使用荧光读板仪通过钙指示剂Fura-2 AM 测定细胞内钙浓度   

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Abstract

Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.

Keywords: Cell biology(细胞生物学), Cell based analysis(基于细胞的分析方法), Ion analysis(离子分析), Calcium(钙), Mammalian cell line(哺乳动物细胞系), Astrocyte(星形胶质细胞), Physiology(生理学)

Background

Activation of G protein coupled receptors triggers production in hundreds or thousands of second messenger molecules. Once stimulated, Gq protein coupled receptors activate phospholipases C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains membrane bound and IP3 is released into the cytosol where it binds to a specific IP3 receptor (calcium channel) in the endoplasmic reticulum (ER). This causes an increase of intracellular calcium concentration that modulates the activation of calcium binding proteins and Protein Kinase C (PKC). Therefore, given the importance of calcium in biological systems, many techniques/methods to analyze and measure Ca2+ levels have been established.
   To determine the intracellular Ca2+ concentration, fluorescent indicators are particularly useful. Ca2+ indicators are available with variations in affinity, brightness or spectral characteristics. Fura-2-acetoxymethyl ester (fura-2 AM), is a membrane-permeable, non-invasive derivative of the ratiometric calcium indicator fura-2. Fura-2 AM crosses cell membranes and once inside the cell, the cellular esterases remove the acetoxymethyl group. Ca2+-bound fura-2 AM has its excitation maximum at 335 nm, Ca2+-free fura-2 AM has its excitation maximum at 363 nm. In both states, the emission maximum is about 510 nm. The typical excitation wavelengths used are 340 nm and 380 nm for Ca2+-bound and Ca2+-free fura-2 AM, respectively. The ratios 510 nm/340 nm and 510 nm/380 nm are directly related to the amount of intracellular Ca2+ (Figure 1). Traditionally, changes in intracellular Ca2+ mobilization/concentration were measured using fluorescent calcium indicators and a fluorescent confocal microscope, known as calcium imaging. This protocol explains how to use the Tecan Infinite M200® microplate reader equipped with an injector to measure intracellular Ca2+ concentration. With the aid of an injector equipped microplate reader, researchers may be able to screen new agonist for Gq protein couple receptors and evaluate how is affected the intracellular calcium mobilization in mutant receptors. This protocol can be adapted to measure agonist mediated intracellular calcium mobilization in several cellular/receptor/agonist models using the appropriate growth conditions.


Figure 1. The human P2Y2 receptor mobilizes intracellular Ca2+ efficiently when expressed in 1321N1 human astrocytoma cells. Representative traces of intracellular calcium responses to 100 μM ATP in 1321N1 cells transfected with hHA-P2Y2R (solid squares) and control non-transfected 1321N1 cells (solid circles). Data represent the mean ± SEM of readings from 4 independent experiments.

Materials and Reagents

  1. 15 ml centrifuge tube (VWR, catalog number: 89004-368 )
  2. Clear flat-bottom black 96-well culture trays with lid (Corning, catalog number: 3904 )
  3. Wild type (WT) human 1321N1 astrocytoma cells devoid of P2 receptors and hHA-P2Y2R expressing 1321N1 cells (gift from Dr. Gary A. Weisman, University of Missouri)
  4. DPBS
  5. Trypsin (TrypLE Express) (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
  6. Probenecid (water soluble) (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36400 )
  7. Adenosine 5’-triphosphate disodium salt (ATP) (Tocris Bioscience, catalog number: 3245 )
  8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  10. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  11. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  12. HEPES (Sigma-Aldrich, catalog number: H0887 )
  13. Glucose (Sigma-Aldrich, catalog number: G8270 )
  14. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  15. Fura-2-acetoxymethyl ester (fura-2 AM) (Thermo Fisher Scientific, InvitrogenTM, catalog number: F1225 )
  16. Pluronic F-127, 20% (w/v) stock in DMSO (Thermo Fisher Scientific, InvitrogenTM, catalog number: P3000MP )
  17. Dulbecco’s modified Eagle’s medium–low glucose (DMEM) (Sigma-Aldrich, catalog number: D2902 )
  18. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
  19. Antibiotic/antimicotic (Sigma-Aldrich, catalog number: A5955 )
  20. Geneticin, G418 (Thermo Fisher Scientific, GibcoTM, catalog number: 10131035 )
  21. HEPES buffered saline (HBS), pH 7.4 (see Recipes)
  22. Dye solution (see Recipes)
  23. Medium for wild type (WT) human 1321N1 astrocytoma cells (see Recipes)
  24. Medium for hHA-P2Y2R expressing 1321N1 cells (see Recipes)
  25. Low serum medium (see Recipes)

Equipment

  1. Tecan Infinite M200® (Tecan Trading, model: Infinite® M200 )
  2. Fisher Scientific Mini vortex (Fisher Scientific)
  3. Eppendorf 5810R Benchtop centrifuge (Eppendorf, model: 5810 R )
  4. Biorad TC10 cell counter® (Bio-Rad Laboratories, model: TC10TM Automated Cell Counter )
    Note: This product has been discontinued.
  5. Fisher Scientific Isotemp CO2 Incubator (5% CO2, 37 °C) (Fisher Scientific)
  6. Laminar flow biological hood

Software

  1. i-Control® (Tecan Trading AG, Switzerland)
  2. Excel® (Microsoft)
  3. GraphPad Prism® (GraphPad Software Inc., La Jolla, CA)

Procedure

  1. Cell culture and fura-2 loading
    1. Maintain cells with the appropriate media in an incubator (37 °C, 5% CO2).
    2. Grow cell to a 90% confluency, discard the cell medium, wash with DPBS and detach with trypsin.
    3. Re-suspend detached cells with 5 ml of cell culture medium and transfer to a 15 ml centrifuge tube.
    4. Centrifuge at 300 x g for 5 min.
    5. Discard cell culture medium and re-suspend cells with 5 ml of low serum medium.
    6. Seed cells 16 h before experiments in clear flat-bottom black 96-well culture trays at a density of 3.0 x 104 cells/well (80-90% confluent).
    7. Discard the medium by carefully inverting the plate and wash once with 200 μl HEPES-buffered saline. Carefully discard HBS by inverting the plate, replace with dye solution and incubate cells 1 h at RT in the dark.
    8. Remove the dye solution by inverting the plate, wash twice with HBS and cover with 200 μl of HBS supplemented with 2.5 mM probenecid for at least 20 min at RT to allow for fura-2 AM de-esterification.
    Note: Cell medium must be without phenol red to avoid fluorescence measurement interference. To avoid evaporation remember to put plate lid back during incubations. 

  2. Instrument setup 
    1. Open the Tecan i-Control software and enter the following parameters:

      Table 1. Dual wavelength Ca2+ indicator assay measurement parameters and instrument settings for the Tecan Infinite M200® with injector. Optimized settings to measure ATP stimulated intracellular Ca2+ mobilization in 1321N1 astrocytoma cells.


    2. Before performing the assay, it is recommended to clean the injectors and tubing first with distilled water, then with HBS.
    3. Prime injector A with 1.1 mM ATP solution.
    4. Set the measurement parameters, insert the plate, remove the plate lid and start the run. Determine fluorescence emission intensity at 510 nm in individual wells alternating excitation wavelengths of 340 and 380 nm every 3 sec for 40 acquisition cycles, using the parameters specified in Table 1.

Data analysis

  1. To express in intracellular calcium concentration in cells, calculate the ratio of 510 nm emission, in response to 340/380 nm excitation.
  2. Normalize Ca2+ mobilization using the average of the first 9 cycles (unstimulated) as baseline.
  3. See Appendix for data processing example.

Notes

  1. Fura-2 AM, under certain staining conditions can accumulate into cell organelles (endoplasmic reticulum, endosome/lysosome, mitochondrion, nucleus). Combinations of dye concentration, incubation time and temperature must be tested to minimize the amount of dye that compartmentalized into organelles (tested by confocal microscopy). Experimental conditions used in this protocol have been optimized for 1321N1 astrocytoma, C6 Glioma and DI TNC cell lines.
  2. Optimal fura-2 loading time and de-esterification time may be determined empirically depending on the cell type. Fura-2 AM, Pluronic F-127, Probenecid concentrations and incubation time stated in this protocol have been optimized for 1321N1 astrocytoma, C6 Glioma and DI TNC cell lines.
  3. Receptor agonist solution preparation:
    1. Agonist solution needs to be prepared taking in consideration well volume and the volume to be dispensed by the instrument injectors.
    2. E.g., Well initial volume = 200 μl
      Volume to be dispense = 20 μl
      Well final volume = 220 μl
      Desired agonist concentration = 100 μM ATP
      Agonist stock concentration = 1.1 mM ATP
  4. Tecan i-Control parameters are optimized for this protocol and must be determined empirically.
  5. HBS can be stored at 4 °C. Allow to reach room temperature before use.
  6. Store Pluronic F-127 20% solution at room temperature. If precipitation is observed, the product may be resolubilized by heating at ~40 °C and vortexing before use.

Recipes

  1. HEPES buffered saline (HBS), pH 7.4
    145 mM NaCl
    5 mM KCl
    1 mM CaCl2
    1 mM MgCl2
    10 mM HEPES
    10 mM glucose
  2. Dye solution
    HEPES buffered saline (HBS), pH 7.4
    0.1% BSA (w/v)
    5 μM fura-2 AM
    0.05% Pluronic F-127
  3. Medium for wild type (WT) human 1321N1 astrocytoma cells
    DMEM
    10% fetal bovine serum (FBS)
    1% antibiotic/antimicotic
  4. Medium for hHA-P2Y2R expressing 1321N1 cells
    DMEM
    10% fetal bovine serum (FBS)
    1% antibiotic/antimicotic
    500 μg/ml Geneticin, G418
  5. Low serum medium
    DMEM
    1% fetal bovine serum (FBS)

Acknowledgments

Partial support to MMA and NAM was received from the Associate Deanship of Biomedical Sciences of the UPR-MSC. Experiments were performed at the Molecular Sciences Research Center of the University of Puerto Rico. 1321N1 astrocytoma cells (WT and P2Y2R expressing) were a gift from Dr. Gary A. Weisman, University of Missouri.

References

  1. Burgos, M., Neary, J. T. and Gonzalez, F. A. (2007). P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells. J Neurochem 103(5): 1785-1800.
  2. Martínez, N. A., Ayala, A. M., Martínez, M., Martínez-Rivera, F. J., Miranda, J. D. and Silva, W. I. (2016). Caveolin-1 regulates the P2Y2 receptor signaling in human 1321N1 astrocytoma cells. J Biol Chem 291(23): 12208-12222.

简介

细胞内钙升高引发广泛的细胞反应。 钙响应可能受到膜受体突变,定位,暴露于激动剂/拮抗剂等的影响或调节(Burgos et al。,2007;Martínez等人,2016年))。 细胞内钙浓度的变化可以使用钙敏感荧光比例染料fura-2 AM测量。 该方法是测量激动剂介导的钙反应的高通量方法。
【背景】G蛋白偶联受体的激活触发了数百或数千个第二信使分子的产生。一旦被刺激,G蛋白偶联受体激活磷脂酶C(PLC),其将磷脂酰肌醇4,5-二磷酸酯(PIP 2 N)切割成二酰基甘油(DAG)和肌醇1 ,4,5-三磷酸酯(IP 3+)。 DAG保持膜结合,并且IP 3被释放到胞质溶胶中,其结合到内质网(ER)中的特异性IP 3'受体(钙通道)。这导致调节钙结合蛋白和蛋白激酶C(PKC)的激活的细胞内钙浓度的增加。因此,考虑到钙在生物系统中的重要性,已经建立了许多分析和测量Ca 2+水平的技术/方法。
 为确定细胞内Ca 2+浓度,荧光指示剂特别有用。 Ca 2 + 指示符可用于亲和力,亮度或光谱特性的变化。 Fura-2-乙酰氧基甲酯(fura-2 AM)是比例计算的钙指示剂fura-2的膜可渗透的非侵入性衍生物。 Fura-2 AM穿过细胞膜,一旦在细胞内,细胞酯酶除去乙酰氧基甲基。 Ca 2 + -bound fura-2 AM在335 nm处具有最大的激发,Ca 2 + - 无fura-2 AM在363 nm处具有最大的激发。在这两种状态下,发射最大值约为510nm。使用的典型激发波长分别为350nm和380nm,分别为Ca 2+ / +和/或不含fura-2 AM。 510nm / 340nm和510nm / 380nm的比例与细胞内Ca 2+的量直接相关(图1)。传统上,使用荧光钙指示剂和称为钙成像的荧光共聚焦显微镜来测量细胞内Ca 2+浓度的移动/浓度的变化。该协议解释了如何使用装有注射器的Tecan Infinite M200 ®微板读数器来测量细胞内Ca 2+浓度。借助于注射器配备的酶标仪,研究人员可能能够筛选G蛋白偶联受体的新激动剂,并评估如何影响突变体受体中的细胞内钙动员。该方案可适用于使用适当的生长条件测量几种细胞/受体/激动剂模型中的激动剂介导的细胞内钙动员。


图1.当在1321N1人星形细胞瘤细胞中表达时,人P2Y2受体有效地动员细胞内Ca 2+。在转染有1321N1细胞的1321N1细胞中100μMATP的细胞内钙应答的代表性痕迹hHA-P2Y 2 R(实心方块)和对照未转染的1321N1细胞(实心圆)。数据表示来自4个独立实验的读数的平均值±SEM。

关键字:细胞生物学, 基于细胞的分析方法, 离子分析, 钙, 哺乳动物细胞系, 星形胶质细胞, 生理学

材料和试剂

  1. 15ml离心管(VWR,目录号:89004-368)
  2. 清洁平底黑色96孔培养托盘盖(康宁,目录号:3904)
  3. 野生型(WT)人类1321N1星形细胞瘤细胞缺乏P2受体和表达1321N1细胞的hHA-P2Y 2 R R(来自密苏里大学Dr.Gary A.Westisman的礼物)
  4. DPBS
  5. 胰蛋白酶(TrypLE Express)(Thermo Fisher Scientific,Gibco TM,目录号:12604013)
  6. 丙磺舒(水溶性)(Thermo Fisher Scientific,Invitrogen TM,目录号:P36400)
  7. 腺苷5'-三磷酸二钠盐(ATP)(Tocris Bioscience,目录号:3245)
  8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  9. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  10. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  11. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  12. HEPES(Sigma-Aldrich,目录号:H0887)
  13. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  14. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  15. Fura-2-乙酰氧基甲酯(fura-2 AM)(Thermo Fisher Scientific,Invitrogen TM,目录号:F1225)
  16. Pluronic F-127,20%(w / v)储备在DMSO中(Thermo Fisher Scientific,Invitrogen TM,目录号:P3000MP)
  17. Dulbecco改良的Eagle中等葡萄糖(DMEM)(Sigma-Aldrich,目录号:D2902)
  18. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178)
  19. 抗生素/抗生素(Sigma-Aldrich,目录号:A5955)
  20. 遗传霉素,G418(Thermo Fisher Scientific,Gibco TM,目录号:10131035)
  21. HEPES缓冲盐水(HBS),pH 7.4(参见食谱)
  22. 染色溶液(参见食谱)
  23. 野生型(WT)人类1321N1星形细胞瘤细胞培养基(见食谱)
  24. 表达1321N1细胞的hHA-P2Y 2 R R的培养基(参见食谱)
  25. 低血清培养基(见食谱)

设备

  1. Tecan Infinite M200 ®(Tecan Trading,型号:Infinite ® M200)
  2. 费雪科学小涡(Fisher Scientific)
  3. Eppendorf 5810R台式离心机(Eppendorf,型号:5810 R)
  4. Biorad TC10细胞计数器(Bio-Rad Laboratories,型号:TC10 TM自动细胞计数器)
    注意:本产品已停产。
  5. Fisher Scientific Isotemp CO 2培养箱(5%CO 2,37℃)(Fisher Scientific)
  6. 层流生物罩

软件

  1. i-Control ®(瑞士Tecan Trading AG)
  2. Excel ®(Microsoft)
  3. GraphPad Prism ®(GraphPad Software Inc.,La Jolla,CA)

程序

  1. 细胞培养和fura-2加载
    1. 在培养箱(37℃,5%CO 2)中用适当培养基保持细胞。
    2. 将细胞生长至90%融合,弃去细胞培养基,用DPBS洗涤并与胰蛋白酶分离。
    3. 用5ml细胞培养基重新悬浮分离的细胞并转移到15ml离心管中。
    4. 以300×g离心5分钟。
    5. 弃去细胞培养液,并用5ml低血清培养基重新悬浮细胞
    6. 在透明的平底黑色96孔培养皿中以3.0×10 4细胞/孔(80-90%汇合)的密度进行实验前16小时的种子细胞。
    7. 通过小心翻转平板并用200μlHEPES缓冲盐水清洗一次,弃去培养基。仔细倒置HBS,更换染料溶液,并在室温下在黑暗中孵育1小时。
    8. 通过翻转板去除染色溶液,用HBS洗涤两次,并用200μl补充有2.5mM丙磺舒的HBS覆盖在室温下至少20分钟以允许fura-2AM去酯化。
    注意:细胞介质必须不含酚红,以避免荧光测量的干扰。为了避免蒸发,请记住在孵化期间将盖板盖回来。 

  2. 仪器设置
    1. 打开Tecan i-Control软件并输入以下参数:

      表1. Tecan Infinite M200的双波长Ca 2 + 指示剂测定参数和仪器设置 ® 与注射器。优化设置以测量ATP刺激1321N1星形细胞瘤细胞中的细胞内Ca 2+活性。


    2. 在进行测定之前,建议先用蒸馏水,然后用HBS清洁注射器和管道。
    3. 主注射器A与1.1mM ATP溶液
    4. 设置测量参数,插入板,取下盖板并开始运行。使用表1中规定的参数,在340个采集周期内,每3秒钟以340和380 nm交替激发波长确定510 nm处的荧光发射强度。

数据分析

  1. 为了在细胞中表达细胞内钙浓度,计算响应于340/380nm激发的510nm发射的比率。
  2. 使用前9个周期的平均值(未刺激)作为基准来归一化Ca 2 + 动员。
  3. 有关数据处理,请参见附录例如

笔记

  1. Fura-2 AM在某些染色条件下可以积累到细胞器(内质网,内体/溶酶体,线粒体,细胞核)中。必须测试染料浓度,孵育时间和温度的组合,以最小化分隔成细胞器的染料的量(通过共聚焦显微镜测试)。本方案中使用的实验条件已针对1321N1星形细胞瘤,C6胶质瘤和DI TNC细胞系进行了优化。
  2. 最佳的fura-2加载时间和去酯化时间可以根据细胞类型凭经验确定。 Fura-2 AM,Pluronic F-127,本方案中阐述的丙磺舒浓度和孵育时间已针对1321N1星形细胞瘤,C6胶质瘤和DI TNC细胞系进行了优化。
  3. 受体激动剂溶液制备:
    1. 激素解决方案需要考虑到体积和仪器注射器要分配的体积。
    2. 初始体积= 200μl
      体积分配= 20μl
      最终体积= 220μl
      所需的激动剂浓度=100μMATP
      激动剂储备浓度= 1.1mM ATP
  4. Tecan i-Control参数针对该协议进行了优化,必须凭经验确定
  5. HBS可以在4°C储存。使用前请保持室温。
  6. 在室温下储存Pluronic F-127 20%溶液。如果观察到沉淀物,则可以通过在〜40℃下加热使产物再次溶解并在使用前涡旋

食谱

  1. HEPES缓冲盐水(HBS),pH 7.4
    145 mM NaCl
    5 mM KCl
    1mM CaCl 2
    1mM MgCl 2
    10 mM HEPES
    10 mM葡萄糖
  2. 染色溶液
    HEPES缓冲盐水(HBS),pH 7.4
    0.1%BSA(w / v)
    5μFfura-2 AM
    0.05%Pluronic F-127
  3. 野生型(WT)人类1321N1星形细胞瘤细胞的培养基 DMEM
    10%胎牛血清(FBS)
    1%抗生素/抗生素
  4. 表达1321N1细胞的hHA-P2Y 2 R的培养基
    DMEM
    10%胎牛血清(FBS)
    1%抗生素/抗生素
    500μg/ ml遗传霉素,G418
  5. 低血清培养基
    DMEM
    1%胎牛血清(FBS)

致谢

从UPR-MSC的生物医学科学副主任收到对MMA和NAM的部分支持。波多黎各大学分子科学研究中心进行实验。 1321N1星形细胞瘤细胞(WT和P2Y 2 R表达)是密苏里大学Gary A. Weisman博士的礼物。

参考

  1. Burgos,M.,Neary,JT和Gonzalez,FA(2007)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/17868308”target = “_blank”> P2Y2核苷酸受体抑制创伤诱导的星形胶质细胞死亡。 J Neurochem 103(5):1785-1800。
  2. Martínez,NA,Ayala,AM,Martínez,M.,Martínez-Rivera,FJ,Miranda,JD and Silva,WI(2016)。  Caveolin-1调节人1321N1星形细胞瘤细胞中的P2Y2受体信号传导。 291(23) :12208-12222。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Martínez, M., Martínez, N. A. and Silva, W. I. (2017). Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader. Bio-protocol 7(14): e2411. DOI: 10.21769/BioProtoc.2411.
  2. Martínez, N. A., Ayala, A. M., Martínez, M., Martínez-Rivera, F. J., Miranda, J. D. and Silva, W. I. (2016). Caveolin-1 regulates the P2Y2 receptor signaling in human 1321N1 astrocytoma cells. J Biol Chem 291(23): 12208-12222.
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