Fluorescence and Bioluminescence Imaging of Subcellular Ca²⁺ in Aged Hippocampal Neurons

The overall goal of this procedure is to investigate calcium remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial calcium in long-term cultures of frat hippocampal neurons, which is considered as a model of neuronal aging. This method can help to understand key questions in the aging field, such as how remodeling of intracellular calcium contributes to excitotoxicity and neuron damage, both related to calcium overload. The main advantage of this technique is that it's possible to follow closely calcium remodeling in individual neurons using cultures at different DYS in vitro from the same specimen duplication of this technique stem through or therapy of a stroke or cell me disease because it variable novel target related to calcium overload in this agent related diseases.

After watching this video, Jesus have a good understanding of how to perform fluorescence and reminiscence imaging of cytosolic and mitochondrial calcium in short term and long-term cultures of rat hippocampal neurons. To begin this procedure, distribute the cover slips on a strip of paraform in a Petri dish. Then apply 200 microliters of poly D lycine at one milligram per milliliter on each cover slip and treat overnight.

The next day wash the cover slips with double distilled sterile water every 15 minutes for 90 minutes under sterile conditions. Place the cover slips in the multi and maintain them in a humidified incubator at 37 degrees Celsius and 5%CO2 until use. To isolate the hippocampal neurons, open the skull of the newborn rat pups with sterile scissors, extract the brain with a spatula and wash it quickly in ham's F 12 medium.

Then make a diagonal cut with the scalpel in each hemisphere and transfer it to the Petri dish containing hams F 12 medium under the microscope. Discard the meninges. Carefully identify the hippocampus in a characteristic concave location over the cortex.

Then separate the hippocampus from the cortex by pulling it away carefully and removing it using eye scissors. Next, transfer the hippocampal pieces to a vial containing 1.8 milliliters of pre-filtered pepane solution. Incubate them at 37 degrees Celsius with occasional gentle shaking for 15 minutes.

Then add 90 microliters of DNA's one solution. Add 50 micrograms per milliliter and incubate them for another 15 minutes. After that, transfer the tissue to a 10 milliliter centrifuge tube and wash the fragments with fresh neuro basal culture medium.

Obtain a cell suspension by passing the tissue fragments through a five milliliter plastic pipette. Next, centrifuge the cell suspension at 160 G for five minutes. Afterward, remove the supinate using a plastic sterile pasture pipette and suspend the pellet carefully in one milliliter of neuro basal culture medium with a one milliliter automated pipette.

Next, measure the cell density using a neubauer counting chamber. Add 10 microliters of cell suspension and count the number of cells under the microscope. Then perform corresponding calculations to obtain 40 to 80 microliters of cell suspension, which contains about 30, 000 cells.

In the four well plate containing 500 microliters of neuro basal medium, played around 30, 000 cells to each well containing glass cover slips. Keep them in the incubator for two to five days in vitro for young neurons or more than 15 days in vitro for aged neurons. In this procedure, incubate the cells with fewer 2:00 AM for 60 minutes at room temperature in the dark.

Then transfer the cover slips to the profusion chamber. After that, place the profusion chamber with the cover slip in the holder on the inverted microscope. Select the microscopic field and perfuse the cells continuously with prewarm HBS.

Subsequently, epi illuminate the cells at 340 and 380 nanometers. Alternately then record the light emitted at 520 nanometers every five to 10 seconds with a fluorescent camera, which is filtered by a fira two diic mirror. To analyze the recorded fluorescent images, first open the experiment file using the Aqua Cosmos software, click on ratio and select the desired ratio range.

Calculate the pixel by pixel ratio in the resulting images in order to obtain a sequence of ratio images. Then subtract the background by adjusting the background elimination button. After that press start calculation.

Next, press the all time sequence button and erase the ancient ROIs for quantitative analysis of individual cells. Establish new ROIs or the ROIs corresponding to the individual neurons. To graph the individual recordings, export the ratio fluorescence values corresponding to each ROI to the Origin lab program by clicking graph.

Then click calculate and save the TXT file. After that, make the corresponding calculation for estimating the size of the rise in the fluorescence ratio in response to each stimulation to transfect cultured neurons with the MIT mutant plasmid. First, prepare 50 microliters of solution A containing transfection reagent in a vial.

Next, prepare 50 microliters of solution B containing MIT ute plasmid in a vial. Add solution B to solution A and incubate for 20 minutes. After that transfer the cover slips containing hippocampal neurons to new multi dishes containing 200 microliters of fresh neuro basal TF.Then add 100 microliters of solution A and B drop by drop over each cover slip and incubate for 30 minutes.

After 30 minutes, remove the medium. Wash the cells with fresh neuro basal tf. Once return the cover slips to the original neuro basal culture medium and culture the cells for 24 hours after transfection to allow efficient expression and targeting of the probe.

Next, add four microliters of sealant TEREZIN N to 200 microliters of HBS to give a final concentration of four micromolar. Mix gently and incubated for two hours at room temperature in the dark. After two hours, transfer cover slips to the perfusion chamber and peruse continuously with prewarm HBS solution.

To record the bioluminescence images of mitochondrial calcium concentration, select the microscopic field containing the cells expressing APO corin as shown by fluorescence emission. Next, turn off the microscope light. Remove the dichroic containing box located in the light pathway.

Then turn off the excitation light and close the dark box for complete darkness. Perfuse the cells with HBS medium, with or without test solutions pre warmed at 37 degrees Celsius. Then capture the photonic emission images every 10 seconds with a photon counting camera handled with an image processor.

Next, add dig toin to release all the remaining photons To analyze bioluminescent images reflecting mitochondrial calcium. Open the GFP fluorescence image. Select individual ROIs by drawing around the transfected cells.

Then copy and paste the same ROIs on every image of the sequence. Screen captures. After that, export the photonic emission values for each ROI to the specific software for calculation click graph.

In order to compute the values for every ROI then select total value and use current ROI to all images. Subsequently, press calculate, save the text file and export the data. Next, make the corresponding calculations for estimating the size of the rises in mitochondrial calcium in response to each stimulus shown.

Here are the representative brightfield and pseudo color images of short-term and long-term cultures of hippocampal neurons before and after stimulation with 100 micromolar and MDA warmer colors reflect elevated cytosolic calcium concentration. These traces show the representative single cell recordings of cytosolic calcium concentration in response to 100 micromolar and MDA in short term and long-term cultures of hippocampal neurons. Note that cytosolic calcium concentration increases are much larger in long term than in short term cultured neurons.

In this experiment, cultured hippocampal neurons were transfected with the low affinity mitochondria targeted korin fused to GFP incubated with four micromolar sealant terezin, and subjected to bioluminescence imaging of mitochondrial calcium concentration. Here, the recordings that show the release of photo emissions reflecting mitochondrial calcium in short and long-term cultured hippocampal neurons. An MDA 100 micromolar increased mitochondrial calcium concentration in aged neurons, but not in young cultured hippocampal neurons Following this procedure.

Other methods like fair left assessment can be combined with fluorescence and bioluminescence imaging in the same cells in order to answer additional questions like susceptibility to neuron cell, lead associated to aging neurodegeneration and ischemia.