Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.
Cell Culture
Cells can be grown using established techniques but must be plated on #1 glass coverslips coated with a cellular adhesive (like polylysine, polyornithine or laminin) to prevent the cells from detaching or moving during imaging experiments.
Solutions
Calcium imaging experiments can be performed using a variety of physiological solutions including cell culture media. It is important, however, to make sure that the solutions are free of phenol red, which greatly increases the fluorescent background. We use Tyrodes solution, which is easily made and mimics cerebrospinal fluid, and we supplement it with 0.1% Bovine Serum Albumin. We use depolarization with 60-90 mM potassium chloride to activate voltage gated calcium channels and 1μM Thapsigargin (1 mM stock in DMSO) or 2μM Ionomycin (1 mM stock in DMSO) to activate store operated CRAC channels. It is often convenient to remove calcium from the extracellular solution to show that calcium elevations are due to calcium influx. When removing calcium it is necessary to maintain the total concentration of divalent cations (Mg2+ and Ca2+) constant. When substituting potassium for sodium it is necessary to maintain the osmotic balance.
Tyrodes solutions:
Low Potassium 2mM Ca2+Tyrodes (mM) | Low Potassium 0 Ca2+ Tyrodes (mM) | High Potassium 2mM Ca2+Tyrodes (mM) | High Potassium 0 Ca2+Tyrodes (mM) | |
NaCl | 129 | 129 | 5 | 5 |
KCl | 5 | 5 | 129 | 129 |
CaCl2 | 2 | 0 | 2 | 0 |
MgCl2 | 1 | 3 | 1 | 3 |
Glucose | 30 | 30 | 30 | 30 |
Hepes | 25 | 25 | 25 | 25 |
Adjust pH to 7.4 with NaOH
Loading of Fura-2 calcium dye
We load cells with acetoxy-methyl-ester Fura-2 (Fura-2 AM), which diffuses across the cell membrane and is de-esterified by cellular esterases to yield Fura-2 free acid. The exact parameters for Fura-2 loading vary widely across cell types. We recommend testing various conditions by preparing several loading solutions containing a multiple concentrations of Fura-2 raging from 1- 4 µM, incubating cells in the loading solution for a variety of times from 15 minutes to 2 hours and testing the loading at room temperature and at 37 deg. A simplified protocol for cortical neurons is given below:
The microscope
We use an inverted Nikon Eclipse TE2000-U microscope equipped with a xenon arc lamp (Sutter Instruments), an automated stage (Ludl), an excitation filter wheel (Ludl), and a cooled charge-couple device (CCD) camera (Hamamatsu Orka II). The microscope is controlled by a Macintosh computer running Open Lab software (Improvision). Several other software packages are available for ratiometric imaging. For imaging we use 40, 60 or 100x Nikon Fluor oil immersion objectives with an NA exceeding 1.2.
Imaging Protocol
Analysis
Once the experiment is complete you will want to convert the set of ratio images into time-lapse calcium measurements for individual cells or regions of interest within cells. To do this:
In this presentation we’ve gone through all the steps for performing calcium imaging using Fura-2 AM. We used cortical neurons as a model but calcium imaging can be used to measure intracellular calcium in real time in a variety of cells. When doing this procedure it is important to use glass coverslips instead of plastic, since plastic is often fluorescent at ultraviolet wavelengths, which makes imaging difficult. Also, it is important to pre-coat the coverslips with polyornithine and laminin in order to prevent cells from detaching. Finally, it’s important to remember to avoid creating air bubbles when perfusing solutions through the imaging chamber.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
New Item | Fura-2 AM | Invitrogen | F1221 | Protect from light |
New Item | DMSO | EM Science | MX1457-6 | Keep in dry place to prevent hydration |
New Item | Albumin from bovine serum | Sigma | A8022-100G | Store at 4° |
New Item | Imaging Chamber | Warner Instruments | Model RC-20H | |
New Item | Microscope | Nikon | Eclipse TE2000-U | |
New Item | Potassium chloride | SIGMA | P9333-500G | |
New Item | Calibration Kit | Molecular Probes | F6774 | Protect from light |