The overall goal of this procedure is to identify new drugs that modulate grk channels. This is accomplished by first plating the A TT 20 cells in the 96 well plate. The second step is to dilute the test compounds using the Verset liquid plate handling system.
Next, the diluted test compounds are applied to the cells using the verset. The final step is to insert the 96 well plate into the Synergy two fluorescent plate reader, and inject the control and somatostatin solutions into the wells. Ultimately, the fluorescent plate reader is used to show the compounds that alter the somatostatin induced fluorescent signal and thus modulate the Grk channels.
The advantage of this procedure over existing methods such as patch clamp recording, is that a large number of compounds can be quickly screened to determine if they modulate gerk channels. The implications of this technique extend towards treatment of pain because compounds that activate the Gerk channel, which lead to inhibition of FI nociceptive transmission, can be identified Demonstrating the procedure for coating. The 96 well plates and plating the cells is Maribel Vasquez.
A student in my laboratory Begin this procedure by growing the pituitary A TT 20 cells in DMEM supplemented with 10%horse serum at 37 degrees Celsius in a humidified atmosphere with 5%CO2. Maintain the culture by sub culturing the cells every five to seven days using a standard ization procedure. Next coat the wells of the black clear.
Bottom 96 well plate with 50 microliters of poly L lysine. Allow the wells to dry for 30 minutes in the incubator. Then remove excess poly L lysine plate the cells in the 96 well plate containing 200 microliters of media at the density of 30, 000 cells per well.
After that store the cells in the incubator for three to four days. Use an aspiration setup to slowly aspirate the culture medium from the cell monolayer, then wash the cells with 200 microliters of normal buffer solution. Subsequently, add 200 microliters of buffer solution containing the membrane potential sensitive dye to each.
Well incubate the cells for 40 minutes at 37 degrees Celsius. After 40 minutes, remove the A TT 20 cells from the incubator and allow them to return to room temperature, aspirate off the old buffer. Then apply fresh buffer solution containing the MPD to the cells.
Next, use the liquid handling system to apply various concentrations of the test compounds and control solutions to the cells. In the 96 well plate incubate the cells for five minutes with test compounds to the fluorescent measurements. In this procedure, insert the 96 well plate into a biotech synergy two fluorescent plate reader.
Obtain the fluorescent background signal at exudation wavelength of 520 nanometers and a mission wavelength of 560 nanometers. Afterward, apply the G PCR R ligands or control solution to the wells to activate the GIR channels using the Synergy two injector system. Then collect the data points at ten second intervals over a 302nd sampling period.
At the excitation and emission wavelengths. Calculate the Z factor in order to determine the reliability of the assay. Z.Factors in the range of 0.5 to 1.0 indicate that the quality of the assay is excellent.
Plates with Z factors below 0.5 are excluded. From the analysis is compounds that modulate the fluorescent signal are retested in the presence of two millimolar barium chloride or 500 nano molar terapin Q to confirm inward rectifier potassium channel specificity shown here is a representative HLB 0 2 1 1 15 2 fluorescent signal measured over time in the A TT 20 cells with the addition of somatostatin or control solution. The ratio of the fluorescent intensity in the Y axis was calculated by dividing the signal in the presence of somatostatin or the control solution by the baseline signal measured before the addition of somatostatin or control solution.
Shown here is the treatment of the A TT 20 cells with 500 nano molars. Terapin Q inhibits the somatostatin mediated decrease in the fluorescent signal by binding two and blocking the G channels. And shown here is the treatment of the A TT 20 cells with 20 micromolar propafenone, which also inhibits the somatostatin mediated decrease in the fluorescent signal Once mastered, this technique can be done in two hours if performed properly.
After viewing this video, you should have a good understanding of how to identify drugs that modulate grk channels using a fluorescent plate reader.
