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Calcium Imaging   

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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.

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)


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


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).


  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)


  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.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Feng, M. (2012). Calcium Imaging. Bio-101: e200. DOI: 10.21769/BioProtoc.200.

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I would like to measure ER Ca2+ Content and therefore use Ca2+ free recording Buffer.
Could you please help me with the question why it is only 'nominally' Ca2+ free and not really 100 % Calcium free?
(why can it not be 100 % Calcium free?) Thank you!
1/19/2019 2:04:24 PM Reply
what kinds of calcium imaging inducers should i use to activate calcium signal?
10/25/2018 5:49:51 AM Reply
Mingye Feng
The Johns Hopkins University School of Medicine

You can use 2 μM thapsigargin (TG) or 2 μM Ionomycin, which depletes ER Ca2+ store and induces Ca2+ influx.

10/25/2018 1:52:17 PM Reply

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 Reply

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 Reply

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