The overall goal of this procedure is to monitor changes in cytosolic calcium concentrations in live cells to differentiate neuronal or glia responses based on potassium chloride, or ATP stimuli. Calcium is an important second messenger involved in several cellular processes, including neurotransmission, plasticity, and apoptosis. The advent of high selective fluorescent calcium dye, such as Fura-2 AM, associated with the development of better fluorescent microscopes and computation methods, yielded high resolution optical data with a high degree of spatial and temporal resolution to image calcium signaling on living cells and organisms.
This protocol is adaptable to enriched neuron, purified glia, or mixed population cultures. It tracks which cell types responded to determinate stimuli based on the differential response to potassium chloride and ATP. Potassium chloride change cell's membrane potential.
And this opens voltage gated calcium channels, expressed essentially by neurons. On the other hand, ATP activates P2X7 large part channel, present mainly in glial cells. By coupling potassium chloride and ATP stimuli with other drugs, it is possible to see which compartment of the nervous system is responding to them, based on the increase of intracellular calcium concentration.
For this procedure, you'll need some surgical instruments or tweezers. Use a biosafety cabinet and perform best practice to avoid contamination. To start the chicken retina culture, carefully open a fertilized egg by the bottom, where the air cell is located.
Remove the contents to a Petri dish and proceed with the embryo donation by decapitation with a pair of tweezers. Once the head is removed, bring it to a clean Petri dish and add some calcium and magnesium free solution to it. Next, remove the eyes, taking care not to damage them in the process.
Bring the eye to another Petri dish containing CMF solution. With a pair of tweezers, begin to dissect the eye by removing the lens. Then, starting by the hole left by the lens, do three or four longitudinal cuts in the eye.
Remove the transparent vitreous body with caution. Make sure the retina is not bind to it. Next, gently detach the retina from the pigmented epithelium.
Remove any remaining epithelial tissue. When the retina is totally clear, cut it in small pieces. Take all the retina tissue with the help of an automated pipette.
Briefly centrifuge the retina to remove all the CMF. Add 1 mL of 0.25%trypsin. And incubate the tissue at 37 C for 10 minutes.
Stop trypsin reaction by adding 1 mL of medium containing 10%fetal calf serum. Centrifuge the retina to remove all the trypsin. Wash the cells two or three times with medium containing 10%FCS.
Next, add 2 mL of medium per retina and gently mechanically dissociate it. Dilute the cells so you can properly count them with an hemocytometer. Use 15 mm coverslips protected with poly-L-lysine and laminin to help cell adhesion.
Pipette 50 L of your diluted cells on each coverslip. Incubate the cells at 37 C in 5%CO2 atmosphere. And chill them to attach the glass.
This should take about one to two hours. Add 1 mL of medium and return it to the incubator until the day of the experiment. If needed change half of the medium every two to three days.
Reconstitute a 50 g vial of Fura-2 AM with 50 L of DMSO. Krebs solution is a good option to transfer cells during the experiment, because it doesn't interfere with fluorescence measurement. Preheat the solution to 37 before beginning.
Add Poloxamer 407. Add Fura-2 AM and DMSO. Sonicate it for seven minutes in a water bath.
Prepare a 6-well plate adding Krebs solution to three wells and the working Fura-2 solution on another. Wash the coverslip contain your cells three times before adding to the Fura-2 well. Incubate the cells at 37 C and 5%CO2 atmosphere for 30 minutes in a dark incubator.
After incubation, wash it three times again. Transfer it to another recipient containing Krebs and protect it from the light. It is possible to reuse the prepared Fura-2 to incubate more coverslips with cells.
Add silicon to the coverslip support and chamber before each run to avoid solution leakage. Take out the coverslip from Krebs solution and carefully wash the bottom with distilled water to avoid salt crystallization on the microscope lens. Put the coverslip on the support, pressing the borders with caution.
Attach the coverslip support and chamber to the microscope, and start perfusing the cells with Krebs solution. Select appropriate cells and choose a good field. Manually determine the regions of interest by selecting cell bodies based on their distinct morphology.
The variations of calcium concentration were evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation at 340 and 380 nm. After adding potassium chloride it's possible to see many cells glowing and the fluorescence response raising on the graph. Potassium chloride opens voltage gated calcium channels, presents mainly in neurons.
Each individual line on the graph represents the unique region of interest. Therefore, it is possible to track individual cell responses during the experiment. ATP stimuli opens P2X7 receptor, essentially expressed by glial cells.
This is an enriched neuron culture. Therefore, there are few glial cells here. This is the reason why so few cells respond to the ATP stimuli.
Acquired values were processed using the Metafluor software. Experimental results are expressed in an Excel table, where each row represent an individual cell, and each line, a time point. Using Excel software, it is possible to plot calcium variations of individual cells separately, or of all of them at the same graph.
To quantify the amount of reactive cells to any stimulus, set a cutoff of 30%increasing calcium baseline levels. Here we use retina cells in culture from embryonic day 8 chicks to investigate how neurons and glia signal in terms of calcium shifts, after receiving 50 mm potassium chloride and 1 mm ATP stimuli. In each experiment, about 100 cells were readily analyzed.
Figure A represents a mixed culture containing both neurons and glia, that was maintained for one week. When we quantify the responses, it is possible to see that about half of the analyzed cells answer potassium chloride, and the other half responded to ATP. Figure B refers to a neuron enriched culture.
It was incubated for only three days, therefore, most glial cells haven't differentiated yet. In this case, 89%of cells answer potassium chloride, reinforcing the prevalence of neurons. Figure C shows a purified glia culture.
These cells were maintained for 10 days with medium changes every three days. After that time most neuron dies, leaving only glia. Indeed, this culture was solely activated by ATP.
This methodology has been used broadly due to the universal properties of calcium as a second messenger. The phenotypical analysis can be enhanced by complimenting this methodology with immunochemistry of fixed cells after calcium imaging. This protocol could also be adapted to other mixed cell systems expressing other calcium channels.
The important tip is to find selective responses of different types of cells correlated with their phenotypic expression.
