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Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry
显微荧光测定腹腔巨噬细胞中的胞内钙浓度   

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Abstract

Calcium is one of the most important intracellular messengers in biological systems. Ca2+ microfluorimetry is a valuable tool to assess information about mechanisms involved in the regulation of intracellular Ca2+ levels in research on cells and in living tissues. In essence, the use of a dye that fluoresces in the presence of a target substance allows the detection of changes in the concentration of this molecule by determining the changes in the fluorescence of the probe (increases or decreases, depending on the nature of the dye used; for a review see Tsien et al. 1985). In this regard, there have been developed two different methodologies to assess intracellular Ca2+ measurements. On the one hand, ratiometric methods are based on the use of a ratio between two fluorescence intensities linked to the physicochemical properties of the probe. This allows correction of artifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated since they require more expensive equipment with the possibility to change the wavelength emission/detection in a rapid way. Some ratiometric Ca2+ indicators are Fura-2 and Indo-1. On the other hand, on binding to Ca2+, indicators used for non-ratiometric measurements show a shift in their fluorescence intensity (the free indicator has usually a very weak fluorescence). Therefore, although an increase in fluorescence signal can be related directly to an increase in Ca2+ concentration, the fluorescence intensity depends on many factors such as acquisition conditions, probe concentration, optical path length, balance between the affinity constants of proteins binding Ca2+, among others. However, the fluxes of Ca2+ are of such a magnitude that these interferences are minor contributors to biases in the measurements. There are many non-ratiometric calcium indicators, some of which are Fluo-3, Fluo-4 and Calcium-Green-3. Consequently, the most suitable Ca2+-probe for each experiment will depend on the range of Ca2+ concentration that has to be evaluated, instrumentation, loading requirements, etc. In the present report we describe the protocol employed to quantify intracellular Ca2+ changes in peritoneal macrophages using Fura-2 as a fluorimetric probe and a microfluorimetric protocol that allows quantification of responding cells to a given stimulus, localization of the main intracellular domains sensing Ca2+ changes and a time-resolved analysis of the Fura-2 fluorescence that reflects the intracellular dynamics of Ca2+ in these cells (Través et al., 2013).

Keywords: Calcium fluxes(细胞内钙流), Myeloid cells(髓系细胞), Single cell analysis(单细胞分析), Microfluorimetry(显微荧光测定法)

Materials and Reagents

  1. Fura 2-AM (Life Technologies)
  2. DMSO (Sigma-Aldrich)
  3. EGTA
  4. CaCl2
  5. HEPES
  6. Trypan Blue
  7. FCS
  8. Vacuum grease
  9. Peritoneal macrophages obtained from Balb/c male mice (8-12 weeks old) 4 days after i.p. administration of 2.5 ml of 3% thioglycollate solution
    Note: In our study (Traves et al., 2013), the effect of prostaglandin E2 on the response to P2X/P2Y agonists ATP and BzATP was studied in depth. All these molecules were purchased from Sigma-Aldrich (St. Louis, MO).
  10. 10x DPBS without Ca2+ Mg2+ (Lonza)
  11. Locke's solution (see Recipes) (supplemented with 1 mg/ml BSA)

Equipment

  1. Coverslips 0.13-0.16 mm thickness (15 mm of diameter) (SCHOTT AG)
  2. Forceps
  3. 35 mm culture dishes
  4. Basic water-jacket CO2 incubator
  5. Perfusion chamber RC-25F (Warner Instruments)
  6. Valves employed for cell perfusion (Warner Instruments)
  7. Perfusion valves controller VC 8 (Warner Instruments)
  8. Vacuum pump
  9. Water bath
  10. NIKON TE-200 microscope with a Plan Fluor x20/0.5 water objective( Nikon Corporation, model:TE-200)
  11. Dichroic mirror (430 nm) and a 510 nm band-pass filter (Omega Optical)
  12. ORCA-ER C4742-80 camera (Hamamatsu Photonics K. K., model: C4742-80)
  13. Filter wheel Lambda 10-2 (Sutter Instrument Company)

Software

  1. MetaFluor 6.2r & PC software (UNIVERSAL SOLUTIONS)

Procedure

  1. Firstly, a sterile coverslip was placed in the bottom of the culture dish (forceps may be helpful) where cells stuck from the very beginning. Macrophages were then carefully seeded in 1 ml of supplemented Locke's solution at a density of 300,000- 500,000 cells per 35 mm culture dish. Since they are non-dividing cells they were used in the following two or three days after seeded (likely, after this lapse time, macrophages will be completely adhered to coverslips). Extensive washing with 500 μl PBS 1X (three-four replicates) was used to ensure that cells were very adherent and alive. The percentage of apoptotic/necrotic cells after 3 days in culture was below 5% (determined by using the common Trypan Blue staining, see Notes).
    Note: Cell attaching to the coverslip is a critical step in order to perform the microfluorimetric analysis. If your plastic or glass interferes with the adherence of the macrophages, allow the support to dry with FCS (fetal calf serum) under the cabin to improve adherence. Avoid a high density cell culture. A 50-75% subconfluent culture with some cells interacting is desirable.
  2. Incubate macrophages in 1 ml supplemented Locke's solution loaded with 5-7 μM Fura-2-AM (dissolved in DMSO, following the datasheet instructions) for 45 min approximately at 37 °C. Incubation does NOT involve either shaking or agitation, just a short and slightly balancing.
  3. Wash thoroughly the culture monolayer with fresh 500 μl Locke's solution (serum also contains esterases that may degrade Fura-2-AM) and place the coverslip in a small superfusion chamber (34 μl volume). Junctions between the coverslip and the chamber are sealed with vacuum grease. Figure 1 depicts the superfusion chamber apparatus.
    Note: Locke’s media or products perfused to the chamber are regulated using a valve system that works by gravity. The perfusion solutions are maintained in a water bath at 37 °C and the flow rate is kept constant at 1.5 ml/min. The vacuum pump aspires continuously the perfusion media after arriving to the chamber to prevent the accumulation of the hydrolysis products.
    Figure 2 shows the experimental setup described here.


    Figure 1. The superfusion chamber system. In the upper side of the figure, we can appreciate the superfusion chamber and the microscope stage. Immediately below these two pictures, both pieces are already assembled. The coverslip should be placed in the circular hollow remaining in the center.


    Figure 2. Experimental equipment for Fura-2-AM registers. The temperature and the valve controllers, the fluorescence microscope (including its optical filter changer) and the necessary equipment to record the fluorescence images (the camera and its controller) are depicted above.

  4. Images of both control and treated cells are visualized using the Plan Fluor x20/0.5 objective of the microscope. In this regard, Fura-2-AM has entered the cell and intracellular esterases have hydrolyzed the compound providing free Fura-2 that senses Ca2+ with a high affinity. Fluorescence emission occurs in a broad range around 430 nm when the cells are excited at 340 nm.
    Note: In our study (Traves et al., 2013), macrophages were preincubated with different prostanoids for at least 10 min and then stimulated for 30 s with a variety of purinergic receptor agonists at near-maximal effective concentrations: 100 μM ATP, 100 μM UTP, 10 μM UDP, 10 μM 2MeSADP, 1,000 μM α,β-meATP or 300 μM BzATP.
  5. Excite cells for 300 ms at 340/380 nm (< 5 ms wavelength change) and select the emitted light using the dichroic mirror (430 nm) and a 510-nm band-pass filter.
    Note: The selection of these wavelengths matches with the maximum fluorescence registers for Fura-2 calcium-saturated solutions (340 nm) and calcium-free Fura-2 solutions (380 nm).
  6. Fluorescence images are acquired with the camera every 1.5 seconds and controlled by the software. Sampling frequency is 2 Hz.

Data analysis

  1. Images are processed by averaging signals from small elliptical regions within individual cells (Figure 3). The possibility exists to define specific areas of changes in the fluorescence emission (cell contact interactions, protrusions in the cytoplasm –cell polarization- or simply cells with different morphologies –round vs. shaped macrophages, depending on the treatments-).
    Note: Background signals are subtracted from each wavelength.


    Figure 3. Metafluor screenshot of cells. ROIs (cells in which a shift in the fluorescence emission is recorded as a function of time) and background (normally areas without cells or non-responsive cells during the period of observation) are marked with a dot in the image. The scale bar on the left side refers to the time variable (in seconds).

  2. The F340/F380 ratio is calculated on the basis of the initial peak magnitude that represents the initial transient components (Figure 4). The F340/F380 ratio is converted into a known calcium concentration using the Grynkiewicz equation:
    [Ca2+] = Kd * (R – Rmin) / (Rmax – R) * F380max/F380min    (Grynkiewicz et al., 1985)
    Where:
    1. Kd is the dissociation constant (depends on the indicator, but also on pH, ionic strength, cell line, etc.).
    2. R is the observed fluorescence ratio at both wavelengths (F340/F380).
    3. Rmin is the minimum ratio value (in absence of Ca2+).
    4. Rmax is the maximum ratio value (when Fura-2 is saturated by Ca2+).
    5. F380max/F380min is a scaling factor (fluorescence intensity at 380 nm excitation in the absence of Ca2+ and at Ca2+ saturation).
      Note: A calibration curve is required to calculate the Kd value. The F380max and F380min values are obtained at the end of each analysis in the same experimental conditions (Fura-2-AM concentration, exposition time…). It is based in two calibration points, a maximum and a minimum corresponding, respectively, to a saturated calcium solution (2.5 mM CaCl2) and a calcium-free solution (containing 10 μM EGTA). Alternatively, inhibition of the membrane reticulum Ca2+ pump with 200 nM thapsigargin or 500 nM ter-buthylbenzohydroquinone allow a saturation of the cytoplasmic Ca2+ concentration. Treatment of cells maintained in extracellular medium lacking or containing Ca2+ with a low dose of ionomycin (ca. 1 μM) to improve the entrance of the Ca2+ into the cell.


      Figure 4. Fluorescence measured at 340/380 nm. A. Fluorescence intensity at both registered excitation wavelengths. B. Registers obtained over the time are divided to determine the F340/F380 ratio.

Notes:

  1. Trypan Blue staining can be used to discriminate between viable and non-viable cells. The protocol follows these typical steps:
    1. Dilute the cell sample (1:10) to a total volume of 20 μl in a 0.4% Trypan Blue dye solution (should be sterile filtered before using).
    2. While non-viable cells will be blue, viable cells will be unstained.
    3. Carefully and continuously fill the hemocytometer chamber with 10 μl of the solution each chamber (all hemocytometers consist of two chambers; each is divided into nine 1mm2 squares).
    4. Count cells under the microscope in four 1 x 1 mm squares of one chamber and determine the average number of cells per square. If the cell density is higher than 200 cells/square, you should dilute your cell suspension.
    5. Total number of particles per ml in the cell sample can be calculated as follows: mean number of cells x 1/dilution factor x 104 cells/ml.

Recipes

  1. Locke's Solution composition
    140 mM NaCl
    4.7 mM KCl
    2.5 mM CaCl2
    1.2 mM KH2PO4
    1.2 mM MgSO4
    5.5 mM glucose
    10 mM HEPES, pH 7.4

Acknowledgments

This work was supported by grants CP11/00080 from ISCIII, BFU2011-024760 from MICINN and FIS-RECAVA RD12/0042/0019. RECAVA and Ciberehd networks are funded by the Carlos III Health Institute. A summary of the procedure was described in Traves et al. (2013).

References

  1. Traves, P. G., Pimentel-Santillana, M., Carrasquero, L. M., Perez-Sen, R., Delicado, E. G., Luque, A., Izquierdo, M., Martin-Sanz, P., Miras-Portugal, M. T. and Bosca, L. (2013). Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J Immunol 190(8): 4226-4235.
  2. Grynkiewicz, G., Poenie, M. and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6): 3440-3450.
  3. Traves, P. G., Pimentel-Santillana, M., Carrasquero, L. M., Perez-Sen, R., Delicado, E. G., Luque, A., Izquierdo, M., Martin-Sanz, P., Miras-Portugal, M. T. and Bosca, L. (2013). Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J Immunol 190(8): 4226-4235.
  4. Tsien, R. Y., Rink, T. J. and Poenie, M. (1985). Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6(1-2): 145-157.

简介

钙是生物系统中最重要的细胞内信使之一。 Ca2 +微量荧光测定法是在细胞和活组织研究中评估涉及细胞内Ca 2+ 2+调节的机制的信息的有价值的工具。本质上,使用在目标物质存在下发荧光的染料允许通过测定探针荧光的变化来检测该分子浓度的变化(增加或减少,取决于染料的性质使用;综述参见Tsien等人.1985)。在这方面,已经开发了两种不同的方法来评估细胞内Ca 2+的测量。一方面,比率法基于使用与探针的物理化学性质相关的两个荧光强度之间的比率。这允许校正由于漂白,焦点变化,激光强度变化等造成的伪影。但是使得测量和数据处理更复杂,因为它们需要更昂贵的设备,具有改变波长发射/检测快速。一些比例的Ca 2+ 2+指示剂是Fura-2和Indo-1。另一方面,在结合Ca 2 + 时,用于非比率测量的指示剂显示其荧光强度的变化(游离指示剂通常具有非常弱的荧光)。因此,虽然荧光信号的增加可以直接与Ca 2+浓度的增加相关,但荧光强度取决于许多因素,例如获取条件,探针浓度,光程长度,蛋白质结合Ca 2+的亲和常数,等等。然而,Ca 2+ 2+的通量具有这样的量级,使得这些干扰对测量中的偏差是较小的贡献。有许多非比例的钙指示剂,其中一些是Fluo-3,Fluo-4和Calcium-Green-3。因此,对于每个实验,最合适的Ca 2+ - 探针将取决于必须评估的Ca 2+浓度的范围,仪器,负载要求, >等。在本报告中,我们描述了使用Fura-2作为荧光探针和微量荧光方案来定量腹膜巨噬细胞的细胞内Ca 2+变化的方案,其允许定量响应细胞对给定的刺激,感测Ca 2+的主要细胞内结构域的定位以及反映Ca 2+ 2的细胞内动力学的Fura-2荧光的时间分辨分析,在这些细胞中(Través等人,2013)。

关键字:细胞内钙流, 髓系细胞, 单细胞分析, 显微荧光测定法

材料和试剂

  1. Fura 2-AM(Life Technologies)
  2. DMSO(Sigma-Aldrich)
  3. EGTA
  4. CaCl <2>
  5. HEPES
  6. 台盼蓝
  7. FCS
  8. 真空润滑脂
  9. 腹膜巨噬细胞从Balb/c雄性小鼠(8-12周龄)腹腔注射后4天获得。 施用2.5ml的3%巯基乙酸溶液
    注意:在我们的研究中(Traves等人,2013),前列腺素E对P2X/T细胞应答的作用, P2Y激动剂ATP和BzATP的深入研究。 所有这些分子购自Sigma-Aldrich(St.Louis,MO)。
  10. 10x DPBS,不含Ca 2+ 2 + Mg 2+ 2 + (Lonza)
  11. 洛克溶液(参见Recipes)(补充有1mg/ml BSA)

设备

  1. 盖片厚度0.13-0.16mm(直径15mm)(SCHOTT AG)
  2. 镊子
  3. 35毫米培养皿
  4. 基础水夹套CO 2子培养箱
  5. 灌注室RC-25F(Warner Instruments)
  6. 用于细胞灌注的阀门(Warner Instruments)
  7. 灌注阀控制器VC 8(华纳仪器)
  8. 真空泵
  9. 水浴
  10. 具有Plan Fluor x20/0.5水物镜(Nikon Corporation,型号:TE-200)的NIKON TE-200显微镜
  11. 二向色镜(430nm)和510nm带通滤光器(Omega Optical)
  12. ORCA-ER C4742-80照相机(Hamamatsu Photonics K.K.,型号:C4742-80)
  13. 滤光片Lambda 10-2(Sutter仪器公司)

软件

  1. MetaFluor 6.2r& PC软件(UNIVERSAL SOLUTIONS)

程序

  1. 首先,将无菌盖玻片置于培养皿的底部(镊子可能是有帮助的),其中细胞从一开始就粘附。然后将巨噬细胞小心地接种在1ml补充的洛克氏溶液中,密度为每35mm培养皿300,000-500,000个细胞。由于它们是非分裂细胞,它们在接种后的两或三天内使用(可能在这段时间之后,巨噬细胞将完全粘附到盖玻片上)。用500μlPBS 1X(三次重复)广泛洗涤以确保细胞非常粘附和存活。培养3天后凋亡/坏死细胞的百分比低于5%(通过使用常见的台盼蓝染色确定,参见注释)。
    注意:连接到盖玻片的细胞是进行微量荧光分析的关键步骤。如果您的塑料或玻璃干扰巨噬细胞的粘附,允许支架用FCS(胎牛血清)在机舱下干燥,以提高粘附。避免高密度细胞培养。需要50-75%的亚融合培养物,其中一些细胞相互作用。
  2. 在加载了5-7μMFura-2-AM(溶解在DMSO中,根据数据表说明书)的1ml补充的Locke's溶液中孵育巨噬细胞,大约在37℃下孵育45分钟。孵育不涉及摇动或搅动,只是短暂和略微平衡
  3. 用新鲜的500μlLocke's溶液(血清还含有可能降解Fura-2-AM的酯酶)彻底洗涤培养物单层,并将盖玻片置于小的表面灌流室(34μl体积)中。盖玻片和腔室之间的接合用真空油脂密封。图1描述了超熔室设备 注意:Locke的介质或灌注到腔室的产品使用通过重力工作的阀系统来调节。将灌注溶液保持在37℃的水浴中,并将流速保持恒定在1.5ml/min。真空泵在到达室后连续地使灌注介质前进,以防止水解产物的累积。
    图2显示了这里描述的实验设置。


    图1.表面灌注室系统。在图的上侧,我们可以欣赏超融合室和显微镜台。紧接在这两张照片的下面,两片都已经组装好了。盖玻片应放在剩余的圆形中空中。


    图2. Fura-2-AM寄存器的实验设备温度和阀门控制器,荧光显微镜(包括其光学过滤器更换器)和记录荧光图像的必要设备(相机和其控制器)如上所述。

  4. 对照和处理的细胞的图像使用显微镜的Plan Fluor×20/0.5物镜可视化。在这方面,Fura-2-AM已经进入细胞,并且细胞内酯酶已经水解该化合物,提供以高亲和力感测Ca 2+ 2 +的游离Fura-2。当细胞在340nm激发时,荧光发射在430nm附近的宽范围内发生 注意:在我们的研究中(Traves等人,2013),巨噬细胞与不同的前列腺素预孵育至少10分钟,然后用各种嘌呤能受体激动剂以接近最大有效浓度刺激30秒:100μM ATP,100μMUTP,10μMUDP,10μM2MeSADP,1,000μMα,β-meATP或300μMBzATP。
  5. 在340/380nm处激发细胞300ms(<5ms波长变化),并使用分色镜(430nm)和510nm带通滤光片选择发射的光。
    注意:这些波长的选择与Fura-2钙饱和溶液(340nm)和无钙Fura-2溶液(380nm)的最大荧光寄存器匹配。
  6. 每1.5秒用相机采集荧光图像,并由软件控制。采样频率为2 Hz。

数据分析

  1. 通过对来自单个细胞内的小椭圆区域的信号进行平均来处理图像(图3)。存在定义荧光发射的改变的特定区域(细胞接触相互作用,在细胞质细胞偏振中的突起,或者简单地具有不同形态的细胞 - 形状巨大的巨噬细胞,取决于处理)的可能性。
    注意:从每个波长中减去背景信号。


    图3.细胞的Metafluor屏幕截图。ROIs(荧光发射的移动被记录为时间的函数的细胞)和背景(通常在没有细胞或非应答细胞的区域观察)在图像中用点标记。左侧的比例尺是指时间变量(以秒为单位)。

  2. F340/F380比率基于表示初始瞬态分量的初始峰值幅度计算(图4)。 使用Grynkiewicz方程将所述F340/F380比率转换为已知的钙浓度:
    [Ca sup 2+] = Kd *(R-R sub)/(R max max)/F max 380+ sub>/F 380min     (Grynkiewicz et al。,1985)
    哪里:
    1. Kd是解离常数(取决于指示剂,但也取决于pH,离子强度,细胞系,等)。
    2. R是在两个波长(F340/F380)处观察到的荧光比率
    3. R min 是最小比值(在没有Ca 2 + 的情况下)。
    4. R submax是最大比率值(当Fura-2被Ca 2饱和时)。
    5. F sub 380/F 380min是比例因子(在没有Ca 2+和Ca 2+的情况下在380nm激发下的荧光强度) 2 + 饱和度)。
      注意:需要校准曲线来计算Kd值。值 在相同实验条件(Fura-2-AM浓度,暴露时间...)的每个分析结束时获得。它基于两个校准点,分别对应于饱和的钙溶液(2.5mM CaCl 2 <2mM )的最大值和最小值,无钙溶液(含有10μMEGTA)。或者,用200nM毒胡萝卜素或500nM叔丁基苯氢醌对膜网膜Ca 2+泵的抑制允许细胞质Ca 2+的饱和,/em> 2 + 集中。用低剂量的离子霉素(约1μM)处理保持在缺乏或含有Ca 2+ 2+的细胞外培养基中的细胞以改善入口的Ca 2 +

      图4.在340/380nm测量的荧光。 A.在两个记录的激发波长下的荧光强度。 B.随时间获得的寄存器被划分以确定F340/F380比率

笔记:

  1. 台盼蓝染色可用于区分存活和非存活细胞。协议遵循以下典型步骤:
    1. 在0.4%台盼蓝染料溶液(应在使用前无菌过滤)中稀释细胞样品(1:10)至总体积为20μl。
    2. 虽然非活细胞将是蓝色的,但活细胞将是未染色的。
    3. 小心地并连续地用10μl的每个室的溶液填充血细胞计数器室(所有血细胞计数器由两个室组成;每个室被分成9个1mm <2μm的正方形)。
    4. 在显微镜下在四个1×1mm方形的一个室中计数细胞,并确定每平方的细胞的平均数量。 如果细胞密度高于200个细胞/平方,你应该稀释你的细胞悬浮液。
    5. 细胞样品中每ml的颗粒总数可以如下计算:细胞平均数×1 /稀释因子×10 4细胞/ml。

食谱

  1. 洛克的解决方案组成
    140mM NaCl 4.7 mM KCl
    2.5mM CaCl 2·h/v 1.2mM KH 2 PO 4 4/
    1.2mM MgSO 4 5.5mM葡萄糖 10mM HEPES,pH7.4

致谢

这项工作得到来自ISCIII的授权CP11/00080,来自MICINN的BFU2011-024760和FIS-RECAVA RD12/0042/0019的支持。 RECAVA和Ciberehd网络由Carlos III健康研究所资助。该程序的概述在Traves等人(2013)中描述。

参考文献

  1. Traves,PG,Pimentel-Santillana,M.,Carrasquero,LM,Perez-Sen,R.,Delicado,EG,Luque,A.,Izquierdo,M.,Martin-Sanz,P.,Miras- Portugal,MT和Bosca ,L.(2013)。 巨噬细胞中前列腺素E2对P2Y信号的选择性损伤:Ca 2 + <> J Immunol 190(8):4226-4235。
  2. Grynkiewicz,G.,Poenie,M。和Tsien,R.Y。(1985)。 新一代的Ca 2 + 指标,具有大大改善的荧光特性。 J Biol Chem 260(6):3440-3450。
  3. Traves,PG,Pimentel-Santillana,M.,Carrasquero,LM,Perez-Sen,R.,Delicado,EG,Luque,A.,Izquierdo,M.,Martin-Sanz,P.,Miras- Portugal,MT和Bosca ,L.(2013)。 通过前列腺素E2在巨噬细胞中对P2Y信号的选择性损伤:对Ca 2+依赖性反应的影响。 em Immunol 190(8):4226-4235。
  4. Tsien,R.Y.,Rink,T.J.and Poenie,M。(1985)。 使用荧光显微镜在单个小细胞中测量胞质游离Ca 2+ 2 + 具有双重激发波长。 Cell Calcium 6(1-2):145-157。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:González-Ramos, S., Carrasquero, L. M., Delicado, E. G., Miras-Portugal, M. T., Fernández-Velasco, M. and Boscá, L. (2013). Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry. Bio-protocol 3(23): e988. DOI: 10.21769/BioProtoc.988.
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Sarbari Acharya
Institute of Life Sciences
Hello

I am measuring the calcium concentration in cancer cells using Fura-2Am. I am unable to understand the Rmin and Rmax values needed to be taken for calibration of the experiment. I am using EGTA (3 uM) and Digitonin (100 um) for RMax and R min respectively. But I am not getting correct values when measuring at 340/380 nm. Could you just clarify the protocol more lucidly.
10/30/2014 1:54:00 AM Reply
María Fernández-Velasco
Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hello,
maybe you can try to calibrate with 2.5 mM CaCl2 (Rmax) and 10 mM EGTA (Rmin). Morover there are some comercial kits to make a calibration curve (check in Invitrogen/Molecular Probes)
María/Lisardo

10/30/2014 8:47:43 AM


Sarbari Acharya
Institute of Life Sciences

Hello

Thanks for your suggestion. But I just wanted to know that whether Rmin and Rmax will be calculated separately or in the same system

10/30/2014 10:59:53 PM


María Fernández-Velasco
Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hello you should calculated R Min and Rmax separately

11/3/2014 1:19:13 AM


Sarbari Acharya
Institute of Life Sciences

Hi

Thanks for the suggestion. Could you tell me what is the incubation time for Rmin and Rmax. Whether the cells should be simultaneously incubated with both EGTA (3 uM) and Fura-2am at the same time. Or first should i pre-incubate cell with EGTA followed by Fura2am loading.

11/5/2014 12:20:20 AM


María Fernández-Velasco
Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hi
You should first incubated cells with FURA and then with EGTA.
Incubation with FURA is 30-40 min.

11/5/2014 4:09:35 AM


Sarbari Acharya
Institute of Life Sciences

Hi

And what about incubation time of EGTA or Cacl2

11/6/2014 10:49:23 PM