The overall goal of this procedure is to demonstrate the application of Flex Station three to study the pharmacological profile of the transient receptor potential or trip a one channel. This is accomplished by first preparing cells that express trip a one channels in 96. Well microtiter plates.
After washing the cells in a microplate washer, the cells are loaded with the calcium sensitive fluorescent dye. Flu oh 4:00 AM a cell loading period is then followed by several additional washes. The next step of the procedure is to prepare the compound plate, including a trip, a one agonist and a putative trip, a one antagonist.
As a final step, the software parameters are set up for plate reading in the Flex Station three system. Ultimately, results can be obtained that show dose response relationships for trip A one activation by flu phonemic acid, and inhibition by the antagonist compound A Though this method can provide insight into 21 activation mechanisms by measuring calcium influx. It can also be applied to study functions of other calcium permeable eye channels and deep protein coupled receptors using calcium sensitive frozen dyes or maly potential dyes.
The main advantage of this Tactic over existing methods like calcium imaging and manual patch mapping is that this time saving technique provides high throughput screening for the function and modulation of calcium formula, iron channels, and other ca mobilizing receptors. Prior to the start of this protocol grow HEC 2 93 cells stably transfected with trip A one to a density of 70 to 90%as described in the written protocol. To begin cell preparation for the calcium assay coat 96 well plates by pipetting 50 microliters of poly L ornithine to each well incubate the plates at 37 degrees Celsius for at least 15 minutes.
Following incubation, rinse the cells once with KO's phosphate buffered saline After trypsin, the HEC 2 93 cells transfer the desired amount of cells to a 15 milliliter sterile conical bottom tube and centrifuge. The sample. Remove the supernatant and resuspend the resulting cell pellet.
In the DMEM culture medium supplemented with 10%heat inactivated fetal bovine serum dispense 100 microliters of the cell suspension to each well of the 96 well plate. Using a multi-channel pipetter, let the plate sit in the hood for at least 30 minutes before placing it in The humidified cell culture incubator for 20 to 44 hours. For calcium assay preparation first prepare solutions as described in the written protocol.
Once prepared, mixed 10 microliters of one millimolar flu oh 4:00 AM with 10 microliters of onic. F1 27, transfer the entire content to five milliliters of flu. Oh four assay buffer supplemented with 0.1%bovine serum albumin.
Next, wash the cells grown in 96. Well plates with HBSS at 80 microliters per well twice using a microplate washer following wash, add the flu oh 4:00 AM loading solution at 50 microliters per well with a multi-channel pipetter and incubate the plate at 37 degrees Celsius for one hour. During the one hour cell loading period, prepare a series of three x solutions of agonists, a series of three x solutions of antagonist, and a large volume of 300 micromolar agonist in the flu oh four assay buffer pipette.
300 microliters of the diluted compound solutions into a 96 well compound plate. Arrange to have the serial dilution of a compound and the corresponding positive and negative controls in the same column of the compound plate whenever possible. As the Flex station three reads one single column at a time, then pipette 300 microliters of the 300 micromolar agonist solution adjacent to each well containing the antagonist as a final cell preparation step.
Wash the loaded cells three times with the flu oh four acid buffer using the microplate washer after the last wash. Leave 80 microliters of the flu oh four acid buffer in each well. To begin plate reading, turn on the Flex Station three system and bring up the soft max Pro 5.2 software.
Save the experiment with a new file name at the setup. Choose flex mode and enter the parameters to measure the agonist and antagonist effect as listed in the written procedure. Next place a new tip box, the compound plate and the reading plate in the appropriate trays of the flex station.Three.
Begin plate reading by clicking the read button in the Soft Max Pro software. The Flex Station three will read one column at a time and add compounds at the pre-programmed time points without interrupting the reading. Once the run is complete, experimental data may be processed directly using the Soft Max PRO software or copied and pasted into any spreadsheet program such as Microsoft Excel.
To analyze the data, generate concentration response curves for the trip, a one agonist flu phonemic acid, and for the putative trip a one antagonist compound a using least squares fitting as described in the written procedure trip. A one is a calcium permeable cation channel activation of which leads to calcium influx that can be detected by the calcium sensitive dye flu oh 4:00 AM.Representative traces of the time courses to fluorescence changes in response to a series of flu phonemic acid or FFA concentrations are shown at higher concentrations. FFA evoked fluorescence increases with a much faster kinetics than at lower concentrations despite similar peak fluorescence levels.
To distinguish these differences, the concentration response relationship was constructed by measuring the initial rates of the fluorescence increase. Upon addition of vary concentrations of FFA, the half maximal effective concentration of FFA was determined to be 55.4 micromolar. The concentration response relationship of inhibition of FFA evoked calcium response by a putative trip a one antagonist compound A is also shown compound.
A dose independently reduced the response of the trip A one cells to FFA with a half maximal inhibitory concentration of 4.3 micromolar. After watching this video, you should have a good understanding of how to study the pharmacological profiles of triple A one and related channels using the flag station three.
