This protocol allows to reveal intercellular calcium signaling pattern that characterizes each cell type response in health as well as in disease. This technique allows fast and detailed biophysical characterization of complex cellular behavior. We plan to use this IgG-induced calcium signaling as a fingerprint for personalized medicine of neuroinflammatory diseases.
In the real life setting, it is not easy to see and follow all the processes and events, especially those happening under the microscope. Hence, the visual data is important. After decapitating one to three-day-old pups, expose the skull by cutting open the skin using small angled scissors.
Then open the skull by cutting the foramen magnum toward the orbits and make a perpendicular midline cut. Remove the brain from the skull, and place it in a Petri dish containing PBS. Use the tip of the forceps to tear the connections between both hemispheres, and separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.
Use straight and curved forceps to remove the meninges by carefully tearing them. Then remove the hippocampus by pinching it with curved forceps, and discard, or use it for another cell culture preparation. Next, transfer the cortex into a 15-milliliter tube containing three milliliters of cold PBS and homogenize the tissue by pipetting up and down 10 to 15 times with a one-milliliter tip.
Centrifuge the cells at 500 g for five minutes. Discard the supernatant and resuspend the pellet in three milliliters of cold PBS by pipetting with a one-milliliter tip. Centrifuge once again, and resuspend the pellet in two milliliters of complete DMEM supplemented with 10%FBS and antibiotics.
After transferring the homogenate to a two-milliliter tube, pass the homogenate through 21 and 23-gauge needles to make a suspension of single cells. Pour the cell suspension prepared from one cortex into a 60-millimeter Petri dish containing three milliliters of complete DMEM, and lightly shake the Petri dish, so that the suspension is uniformly distributed. Incubate the cells in a humidified incubator with 5%carbon dioxide and 95%air at 37 degrees Celsius.
To promote astrocyte growth, remove the old media, and wash the cell with pre-warmed PBS to remove the loosely attached glial cells and traces of FBS once they attain 70 to 80%confluency. Then trypsinize the underlying layer of astrocytes by adding one milliliter of pre-warmed trypsin solution and incubate at 37 degrees Celsius for two to five minutes. Use a microscope to check if the cells begin to detach, and add four milliliters of complete DMEM.
Collect the cell suspension, and transfer it to a 15-milliliter tube. Then centrifuge the tube at 500 g for five minutes. Discard the supernatant and resuspend the pellet in one milliliter of complete medium.
Count the cells using a hemocytometer. Next, prepare the culture dish and add culture medium. Replate the cells at a density of 10 to the fourth cells per square centimeter in five milliliters of fresh complete DMEM and wash the cells with complete DMEM before each media replacement.
After reaching 70 to 80%confluency, repeat trypsinization, and seed five times 10 to the third astrocytes on a seven-millimeter poly-L-lysine-coated circular glass cover slip. Let them attach for 10 minutes, and add complete DMEM. Use them in experiments after 48 hours.
After preparing the microglial cultures, to promote microglia, allow the glial cells to become confluent and the microglia will appear on top of the astrocyte layer after 10 to 15 days. Shake the Petri dish on an orbital shaker for two hours at 220 RPM. Now, lightly wash the detached and loosely attached cells by aspirating the supernatant with a one-milliliter pipette tip.
Gently dispense the supernatant onto the layer of cells several times, ensuring to cover the whole cell layer surface during this washing step. Collect the medium with the detached cells, and transfer it to a 15-milliliter tube. Centrifuge, resuspend, and count the cells as demonstrated earlier.
Seed five times 10 to the third microglia on a seven-millimeter poly-L-lysine-coated circular glass cover slip. Let them attach for 10 minutes and add complete DMEM. Use them in experiments after 48 hours.
To wash out the complete DMEM, transfer one cover slip to a dish with extracellular solution. Prepare the dye loading solution by diluting one millimolar Fluo-4 AM with extracellular solution to achieve the final concentration of five micromolar. Place the cover slip with cells in the dye loading solution for 30 minutes at room temperature in the dark, and wash the cells in extracellular solution for 20 minutes.
Place the cover slip in the recording chamber with one milliliter of working solution. Choose the field of view having a consistent number of cells throughout the experiment. Start the imaging and determine the baseline by acquiring the basal level of fluorescence for three to five minutes.
Then stop the flow of the working solution, and switch to the test solution for the desired length of time. Between each test solution, wash the cells with a constant flow of the working solution for three to five minutes. Keep the solution volume in the recording chamber at one milliliter by arranging a constant suction from the top of the solution.
Choose the highest signal intensity frame, and encircle one cell using the Polygonal tool application. After encircling all the cells in the acquired field, choose five regions of interest in the background using the Circle tool. Then measure the mean signal intensity of a single cell and the background for each timeframe.
Select all regions of interest, and use the Multi-Measure command in ROI Manager in ImageJ, or any equivalent command in commercial software. After importing the data in MATLAB software, average five background ROIs and subtract the averaged background of each frame from the mean ROI intensity of the frames acquired simultaneously. Later, analyze the calcium activity by determining the amplitude of the calcium peak, the integrated change of the calcium transient, time to peak, rise time, half-width, and frequency.
GFAP is used to visualize astrocytes and Iba1 to visualize microglia. hSOD1G93A astrocytes respond to ALS immunoglobulin G with greater amplitude of the calcium transient, a more considerable overall integrated change, and a shorter time to peak than non-transgenic astrocytes. The response to ALS immunoglobulin G is distinguishable from the response to ATP.
hSOD1G93A astrocytes had a higher level of calcium ions in the stores as revealed by store depletion, indicating an overload of this ion. Microglia were cultured, and the mean fluorescence intensity was extracted over time. The calcium traces of three cells represented that only one cell responded to ALS immunoglobulin G, while all cells responded to ATP.
The pseudo-color images represented the fluorescence intensity and baseline in response to ALS, IgG, and ATP. The cells need to be adequately loaded and the parameters of the imaging system need to be appropriately adjusted, so that the signal is not separated during the experiment. Along with calcium-emitting, patch clamping can be performed.
Thus, a more complete picture of the correlation between different membrane ion currents and calcium-emitting can be obtained. The understanding of intracellular calcium homeostasis is of essential importance for the exploration of several response to natural as well as to stressful and external stimuli.
