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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.

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[Bio101] Calcium Imaging

Cell Biology > Cell-based analysis > Ion analysis > Calcium
Author: Mingye Feng
Mingye FengAffiliation: Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: mfeng@jhmi.edu
Bio-protocol author page: a47
4/5/2012, 17594 views, 4 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.200

[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.         NaCl

3.         MgCl2

4.         KCl

5.         Glucose

6.         HEPES

7.         CaCl2

8.         PBS

9.         Fura-2-AM (Invitrogen, F1221)

10.     Poly-L-Lysine (Sigma, P8920)

11.     Fura-2 calcium imaging calibration kit (Invitrogen, F6774)

 

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 thecoverslips with 0.01% poly-L-Lysine (dilute the 0.1% stock as 1:10 with sterilized water) at RT for 10 minutes and washed three times with sterilized PBS.

2)       Plate HEK293 cells on coverslips 24 hours before Ca2+ imaging. If cells need to be transfectedwith GFP reporter, seed cells 48 hours before Ca2+ imaging and transfect cells 24 hours 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 minutes each.

2)       Dissolve the cell-permeant acetoxymethyl ester of the calcium indicator Fura-2 (Fura-2-AM, 50μg /vial) in 50μl of DMSO and then dilute into Ca2+ recording buffer.

3)       Load HEK293 cells with 1 μg /ml Fura-2-AM at RT for 30 minutes (Fura-2-AM can be used for up to 5 μg/ml for different types of cells). Wash cells twice with recording buffer, 5 minutes each time.

3.         Prepare perfusion system (Smartsquirt small volume delivery system)

1)       Turn on the nitrogen tank with pressure up to 10psi-20psi.

2)       Turn on vacuum pump.

3)       Wash the buffer reservoir.

4)       Adjust the pressure regulator to reach a 2ml/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 0.5ml 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 510nm;

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 seconds); Exposure time for 340nm (typically 100ms); Exposure time for 340nm (typically 20ms). Parameters can be adjusted according to experiment design and cells to be measured.

4)       Click the “W1” icon to expose cells to 340nm; adjust the focus plane to get the best image; adjust the exposure time to control the intensity to around 1000 (the maxi value is 4999).

5)       Click the “W2” icon to expose cells to 380nm; adjust the exposure time to control the intensity to around 1000 (the maxi value is 4999).

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 340nm and 380nm every 3 seconds. The ratios of 340/380 nm indicate the intracellular Ca2+ concentration.

6.         Export data to Excel

1)       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 2mM 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 0Ca 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 (0.5mM, 1mM, 2mM et al.). Keep recording intracellular Ca2+concentration until it reaches the plateau (typicall 3-5 minutes) (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 minutes) (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 0Ca recording buffer with 2mM EDTA or treating cells with 10μM Ionomycin in recording buffer containing 10mM Ca2+. F380max and F380min are the fluorescence intensity of 380nm excitation at 0Ca 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., Rao R. (2010).Store-independent activation of orai1 by spca2 in mammary tumors. Cell 143(1): 84-98.

2.       Grynkiewicz G., Poenie M., Tsien R.Y. (1985). A new generation of ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260(6): 3440-50. 

 

 

 

 



How to cite this protocol: Feng, M. (2012). Calcium Imaging. Bio-protocol Bio101: e200. DOI: 10.21769/BioProtoc.200; Full Text



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5/12/2014 2:17:04 AM  

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?

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4/25/2012 4:03:51 AM  

The emission wavelength of Fura-2 and GFP are overlapped , not the excitation wavelength

4/25/2012 1:15:25 PM  

MIngye Feng (Author)
Department of Physiology,The Johns Hopkins University School of Medicine

Hope the answer posted below have answered your question.

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4/25/2012 1:34:32 AM  

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 1:10:16 PM  

MIngye Feng (Author)
Department of Physiology,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.

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3/31/2012 6:05:14 AM  

记录到的数值是单个细胞还是一组细胞?可否只记录单个细胞?

3/31/2012 11:09:32 PM  

MIngye Feng (Author)
Department of Physiology,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.

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