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[Bio101] Calcium Imaging
[Bio101] 钙成像   

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Abstract

This is a protocol of Ca2+ imaging experiment using Ca2+ indicator Fura-2. Ca2+ imaging is an efficient and quantitative method for measuring cytosolic and internal store Ca2+ levels, as well as their dynamic changes.

Materials and Reagents

  1. HEK293 cells
  2. Phosphate buffered saline (PBS)
  3. Fura-2-AM (Life Technologies, Invitrogen™, catalog number: F1221 )
  4. Poly-L-lysine (Sigma-Aldrich, catalog number: P8920 )
  5. Fura-2 calcium imaging calibration kit (Life Technologies, Invitrogen™, catalog number: F6774 )
  6. NaCl
  7. MgCl2
  8. KCl
  9. Glucose
  10. HEPES
  11. CaCl2
  12. DMSO
  13. GFP reporter
  14. Lonomycin
  15. Ca2+ recording buffer (see Recipes)

Equipments

  1. Zeiss inverted microscopy with perfusion system and “IPlab” software.
  2. Centrifuges

Procedure

Note: Target cells: concentration of Fura-2-AM may need to be optimized depending on cell types to be measured. The following protocols are designed for HEK293 cells.

  1. Prepare the cells
    1. Autoclaved coverslips or multi-well coverglass chambers are used for culturing cells. Coat the coverslips with 0.01% poly-L-lysine (dilute the 0.1% stock as 1:10 with sterilized water) at RT for 10 min and washed three times with sterilized PBS. 
    2. Plate HEK293 cells on coverslips 24 h before Ca2+ imaging. If cells need to be transfected with GFP reporter, seed cells 48 h before Ca2+ imaging and transfect cells 24 h after seeding. For coverslips in 6-well plates, 0.2-0.5 millions of cells should be seeded to each well. Numbers of the cells to be seeded can be adjusted according to the size of plate/chamber used.

  2. Load the cells
    1. Wash cells with Ca2+ recording buffer twice at RT, 5 min each. 
    2. Dissolve the cell-permeant acetoxymethyl ester of the calcium indicator Fura-2 (Fura-2AM, 50 μg/vial) in 50 μl of DMSO and then dilute into Ca2+ recording buffer.
    3. Load HEK293 cells with 1 μg/ml Fura-2-AM at RT for 30 min (Fura-2-AM can be used for up to 5 μg/ml for different types of cells). Wash cells twice with recording buffer, 5 min each time.

  3. Prepare perfusion system (Smartsquirt small volume delivery system)
    1. Turn on the nitrogen tank with pressure up to 10 psi-20 psi.
    2. Turn on vacuum pump.
    3. Wash the buffer reservoir.
    4. Adjust the pressure regulator to reach a 2 ml/min flow speed.
    5. Load the buffer reservoir with Ca2+ recording buffer.

  4. Load coverslips onto microscopy
    1. Place the coverslip in a perfusion chamber and load with 0.5 ml of Ca2+ recording buffer.
    2. Turn on the Zeiss inverted microscopy; mount the perfusion chamber on the stage of microscopy; use 40x oil emersion lens;
    3. Turn on the power of Zeiss FluorArc mercury lamp, and turn the filter to “Fura-2” which will collect the emission response at 510 nm;

  5. IPlab software
    1. Turn on the camera connecting to microscopy;
    2. In IPlab, click the ratiometric icon, set the parameters:
    3. Total time points; Interval time (typically 1-10 sec); Exposure time for 340 nm (typically 100 ms); Exposure time for 340 nm (typically 20 ms). Parameters can be adjusted according to experiment design and cells to be measured.
    4. Click the “W1” icon to expose cells to 340 nm; adjust the focus plane to get the best image; adjust the exposure time to control the intensity to around 1,000 (the maxi value is 4,999).
    5. Click the “W2” icon to expose cells to 380 nm; adjust the exposure time to control the intensity to around 1,000 (the maxi value is 4,999).
    6. Select the individual cells to be measured as well as the background region. Multiple cells can be selected in each field.
    7. Start recording, image will be acquired by exciting the cells at 340 nm and 380 nm every 3 sec. The ratios of 340/380 nm indicate the intracellular Ca2+ concentration. 

  6. Export data to excel
    In IPlab, open the ratio-plot file, from “view” - “new” - “text”, export data as excel files.

  7. Applications
    1. Measure ER Ca2+ content
      For the experiments measuring ER Ca2+ content in HEK293 cells, cells were switched from 2 mM Ca2+ to nominally Ca2+ free (0 mM Ca2+) recording buffer by using the perfusion system, and 2 μM of ionomycin was added to deplete Ca2+ stores (Feng et al., 2010). 
    2. Measure Ca2+ influx and efflux
      For the experiments measuring Ca2+ influx and efflux, incubate HEK293 cells in 0 Ca recording buffer during Fura-2 loading. Measure basal Ca2+ level for 10-20 time points to make sure Ca2+ level has become stable. Carefully remove recording buffer from the perfusion chamber and rapidly add back recording buffer with different Ca2+ concentrations (0.5 mM, 1 mM, 2 mM et al.). Keep recording intracellular Ca2+ concentration until it reaches the plateau (typicall 3-5 min) (the increase of intracellular Ca2+ concentration indicates Ca2+ influx). Carefully remove recording buffer from the perfusion chamber and rapidly add back 0 Ca recording buffer. Keep recording intracellular Ca2+ concentration until it reaches the plateau (typicall 3-5 min) (the decrease of intracellular Ca2+ concentration indicates Ca2+ eflux) (Feng et al., 2010). 
    3. Calculation of Ca2+ concentration
      Ca2+ concentration can be calculated by:
      [Ca2+] = Kd * (R – Rmin) / (Rmax – R) * F380max / F380min (Grynkiewicz et al., 1985)
      R is the measured 340/380 nm ratio; Rmin and Rmax are the ratios in absence of Ca2+ or when Fura-2 is saturated by Ca2+, and can be determined by incubating cells in 0 Ca recording buffer with 2 mM EDTA or treating cells with 10 μM Ionomycin in recording buffer containing 10 mM Ca2+. F380max and F380min are the fluorescence intensity of 380 nm excitation at 0 Ca and Ca saturation. Kd can be calibrated using the calibration kit from Invitrogen (F-6774).

Recipes

  1. Calcium recording buffer
    125 mM NaCl
    2 mM MgCl2
    4.5 mM KCl
    10 mM Glucose
    20 mM HEPES pH 7.4
    2 mM CaCl2 (no CaCl2 was added for the 0 Ca2+ buffer)

References

  1. Feng, M., Grice, D. M., Faddy, H. M., Nguyen, N., Leitch, S., Wang, Y., Muend, S., Kenny, P. A., Sukumar, S., Roberts-Thomson, S. J., Monteith, G. R. and Rao, R. (2010). Storeindependent activation of orai1 by spca2 in mammary tumors. Cell 143(1): 84-98.
  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.

简介

这是一个利用Ca2+指示剂Fura-2的钙成像实验步骤。钙成像是一个有效的定量的方法测量细胞质内,和内部的Ca2+水平,以及他们的动态变化。

材料和试剂

  1. HEK293细胞
  2. 磷酸盐缓冲盐水(PBS)
  3. Fura-2-AM(Life Technologies,Invitrogen TM,目录号:F1221)
  4. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P8920)
  5. Fura-2钙成像校准试剂盒(Life Technologies,Invitrogen TM,目录号:F6774)
  6. NaCl
  7. MgCl 2
  8. KCl
  9. 葡萄糖
  10. HEPES
  11. 氯化钙<子> 2
  12. DMSO
  13. GFP记者
  14. 离子霉素
  15. 记录缓冲区(见配方)

设备

  1. 蔡司倒置显微镜与灌注系统和“IPlab”软件
  2. 离心机

程序

注意:靶细胞:Fura-2-AM的浓度可能需要根据要测量的细胞类型进行优化。以下协议是为HEK293细胞设计的

  1. 准备细胞
    1. 高压灭菌盖玻片或多孔盖玻璃室用于培养细胞。将盖玻片用0.01%聚-L-赖氨酸(用灭菌水稀释0.1%原料,1:10)涂覆10分钟,用无菌PBS洗涤3次。
    2. 在Ca 2 + 成像前24小时,在盖玻片上铺板HEK293细胞。如果细胞需要用GFP报告基因转染,种子细胞在Ca2 +成像48小时后和播种24小时后转染细胞。对于6孔板中的盖玻片,应将0.2-0.5百万个细胞接种到每个孔中。可以根据所使用的板/室的尺寸来调整要种植的细胞的数量。

  2. 加载单元格
    1. 用Ca 2 + 记录缓冲液在室温下洗涤两次,每次5分钟。
    2. 将钙指示剂Fura-2(Fura-2AM,50μg/小瓶)的细胞透过性乙酰氧甲基溶于50μlDMSO中,然后稀释成Ca 2+。记录缓冲液。
    3. 在室温下加入含有1μg/ ml Fura-2-AM的HEK293细胞30分钟(对于不同类型的细胞,Fura-2-AM可以用于高达5μg/ ml)。用记录缓冲液洗涤细胞两次,每次5分钟。

  3. 准备灌注系统(Smartsquirt&nbsp;小批量交付系统)
    1. 打开氮气罐,压力高达10 psi-20 psi。
    2. 打开真空泵。
    3. 洗涤缓冲液储存器。
    4. 调节压力调节器达到2 ml / min的流速。
    5. 用Ca 2 + 记录缓冲液装载缓冲液储存器。

  4. 将盖玻片加载到显微镜上
    1. 将盖玻片置于灌注室中,并加入0.5ml Ca 2 O 3 +记录缓冲液。
    2. 打开蔡司倒置显微镜;将灌注室安装在显微镜的阶段;使用40x油浸透镜;
    3. 打开Zeiss FluorArc水银灯的电源,并将滤光片转到“Fura-2”,将收集510 nm的发射响应;

  5. IPlab软件
    1. 打开连接到显微镜的相机;
    2. 在IPlab中,单击比例图标,设置参数:
    3. 总时间间隔时间(通常为1-10秒); 340 nm的曝光时间(通常为100 ms); 340 nm的曝光时间(通常为20 ms)。参数可以根据实验设计和要测量的单元进行调整。
    4. 单击“W1”图标将单元格曝光到340 nm;调整焦点平面以获得最佳图像;调整曝光时间以将强度控制在1,000左右(最大值为4,999)。
    5. 单击“W2”图标将单元格暴露于380 nm;调整曝光时间以将强度控制在1,000左右(最大值为4,999)。
    6. 选择要测量的单个单元格以及背景区域。可以在每个字段中选择多个单元格。
    7. 开始记录,通过每3秒激发340nm和380nm的单元将获得图像。 340/380 nm的比值表示细胞内Ca 2+浓度。

  6. 导出数据到excel
    在IPlab中,打开比例图文件,从“视图” - “新” - “文本”中导出数据为Excel文件。

  7. 应用
    1. 测量ER Ca 2 + 内容
      对于测量HEK293细胞中的ER Ca 2+ 2 + 含量的实验,将细胞从2mM Ca 2+升高至标称Ca 2+, 0mM Ca 2+ 2 + )记录缓冲液,加入2μM的离子霉素以消耗Ca 2+ 。,2010)。&nbsp;
    2. 测量Ca 2 + 流入和流出
      对于测量Ca 2 + 流入和流出的实验,在Fura-2加载期间将0KC记录缓冲液中的HEK293细胞孵育。测量基础Ca 2 + 水平10-20个时间点,以确保Ca 2 + 水平变得稳定。小心地从灌注室中取出记录缓冲液,并快速添加具有不同Ca 2+浓度(0.5mM,1mM,2mM等)的记录缓冲液。保持记录细胞内Ca 2+浓度直到达到平台(典型的3-5分钟)(细胞内Ca 2+浓度增加浓度表示Ca 2+ influc)。小心地从灌注室中取出记录缓冲液,并快速添加0 Ca记录缓冲液。保持记录细胞内Ca 2+浓度直到达到平台(典型的3-5分钟)(细胞内Ca 2+浓度的降低表明Ca 2+浓度 eflux)(Feng等人,2010)。
    3. 计算Ca 2 + 浓度
      Ca 2 + 浓度可以通过以下公式计算:
      [Ca 2 + ] = Kd *(R-R min min)/(R max max -R)* F 380max /> 380分钟(Grynkiewicz等人,1985)
      R是测量的340/380 nm比例; Rmin和Rmax是不存在Ca 2+ / +或当Fura-2被Ca 2+ 2 +饱和时的比例,并且可以通过在0Ca记录缓冲液中孵育细胞来确定用2mM EDTA或用10μM离子霉素处理含有10mM Ca 2+的记录缓冲液中的细胞。 F 380nm和F 380min是0 Ca和Ca饱和度下380nm激发的荧光强度。可以使用Invitrogen(F-6774)的校准试剂盒对Kd进行校准。&nbsp;&nbsp;&nbsp;

食谱

  1. 钙记录缓冲液
    125 mM NaCl
    2mM MgCl 2
    4.5 mM KCl
    10 mM葡萄糖
    20mM HEPES pH 7.4
    对于0 Ca 2+缓冲液,加入2mM CaCl 2(不含CaCl 2)

参考

  1. Feng,M.,Grice,DM,Faddy,HM,Nguyen,N.,Leitch,S.,Wang,Y.,Muend,S.,Kenny,PA,Sukumar,S.,Roberts-Thomson,SJ,Monteith, GR and Rao,R。(2010)。&nbsp; 存储独立激活的乳腺肿瘤中的spca2的orai1。 143(1):84-98。
  2. Grynkiewicz,G.,Poenie,M.和Tsien,RY(1985)。&nbsp; 新一代具有极大改善的荧光性质的Ca2 +指示物。 260(6):3440-3450。
  • English
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Feng, M. (2012). Calcium Imaging. Bio-protocol Bio101: e200. DOI: 10.21769/BioProtoc.200;
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Chelsea Mark
Can you give more detail about How to make Calcium recording buffer??? How much calcium recording buffer (ml) did you need for this experiment at least?
5/12/2014 2:17:04 AM Reply
The emission wavelength of Fura-2 and GFP are overlapped , not the excitation wavelength
4/25/2012 4:03:51 AM Reply
Mingye Feng
The Johns Hopkins University School of Medicine

Hope the answer posted below have answered your question.

4/25/2012 1:15:25 PM


The excitation wavelength of Fura-2 is overlapped with GFP. If the cells to be measured express GFP, does this affect the measurement? what is the difference between Fura-2(Calcium free) and Fura-2(Calcium bound)?
4/25/2012 1:34:32 AM Reply
Mingye Feng
The Johns Hopkins University School of Medicine

GFP should have minor effects on Fura-2 measurement, because its major excitation peak is over 450nm, while Fura-2 is excited at 340 and 380nm. But a GFP-only control would be necessary for the measurement.

Fura-2(Calcium free) and Fura-2(Calcium bound) have different excitation wavelength. When Fura-2 is bound to calcium, its excitation peak is 340nm. When Fura-2 is not bound to calcium, it will be 380nm.

4/25/2012 1:10:16 PM


3/31/2012 6:05:14 AM Reply
Mingye Feng
The Johns Hopkins University School of Medicine

Yes, this protocol can be used to measure the Ca 2+ concentration for a single cell. Please refer to step 5 - IP lab - 6). Due to the variations between individual cells, it's recommended to measure multiple cells and get the average.

3/31/2012 11:09:32 PM