The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.
This method can help the discovery of NMDA modulators to treat neurological diseases. The main advantage of this technique is that it allows the fissum study of NMDA-receptor activity, in response to defense concentrations or combinations of ligands in compounds. So this method can provide insight into the general capability of NMDA receptor.
It can also be applied to study subtypes specific in biology and a sensitivity of NMDA receptor, in response to ligands or compounds. To measure the activity of the NMDA-receptor, prepare a 384 well plate, with HEK 293 cells, transduced with NR1, NR2, or HEK 293, NR1, NR2A cells in the presence of protective compound. Then, incubate the plate at 37 degrees Celsius, and five percent carbon dioxide for 16 hours.
The next day, remove the plate from the incubator, and gently discard the cell media, by slowly flipping the plate over a compatible biowaste container. Gently tap the plate on a paper towel, to clean the media off the edges of the plate, and drain any remaining media. Subsequently, prepare the calcium 6 dye, by solubilizing one vial of calcium 6 dye, in 10 milliliters of incubation buffer.
Add 30 microliters of the dye to the cells in the 384 well plate. Incubate the plate for two hours at 37 degrees Celsius, and five percent carbon dioxide. After two hours, remove the plate from the incubator, and let the cells adjust to room temperature for 10 minutes.
Next, gently pour out the calcium 6 dye, and add 30 microliters of assay buffer. Repeat the wash for a total of three times, and leave 30 microliters of assay buffer on the cells, after the last wash. Then, let the cells rest for five minutes at room temperature.
To prepare the ligand plate, make a four-fold stock of 400 micromolar glycine, and 400 micromolar glutamate. Transfer a minimum of 25 microliters of ligand stock into a V Bottom 384 well plate. Then transfer the ligand plate and cell plate into the FDSS.
Afterward, measure the baseline fluorescence of the cell plate for 30 seconds. Transfer 10 microliters of the ligand stock into the cell plate, using the FDSS dispensing function, and measure the calcium-flux by recording calcium 6 dye fluorescence for five minutes. Afterward, calculate the maximal fluorescence ratio, by dividing the maximal fluorescence obtained by the baseline fluorescence.
To test the compounds, prepare the cells, the ligand, and the compound plates. Load the ligand, compound, and cell plate into the FDSS. Next, measure the baseline fluorescence for 30 seconds.
Add 10 microliters of compound stock, and measure the fluorescence for five minutes. Then, add 10 microliters of ligand stock, and measure the fluorescence for five minutes. Afterward, determine the integral of the fluorescence ratio by calculating the area under the curve of the fluorescent ratios during the last five minutes of the measurement.
Using the predetermined optimal ligand and subunit expressions, the effects of a glutamate binding site antagonist, NVP-AAM077, and the glycine binding site antagonist, L701, 324, were characterized by the calcium-flux assay, for NR1, NR2A at the indicated concentrations. A concentration equal to the eZ80 of glycine or glutamate, in the presence of the saturating concentration of the other ligand, was used after addition of NVP-AAM077, and L701, 324, respectively. While attempting this procedure, it's important to remember to optimize the respective various amounts for this specific cell lines, using the experiment.
And to use healthy nonconfluent cells. Special occasion should be spent on the dilution of compounds and ligands. Following this procedure, other methods like electrophysiology can be performed, in order to assign additional questions about a specific mechanism of actions, or their compounds.
