The overall goal of this procedure is to discover novel compounds that modulate the activity of inward rectifier potassium channels. This is accomplished by first plating tetracycline inducible, HK 2 9 3 cells, expressing a KIR channel of interest into a 384 well plate. The second step is to load the cells with a fluorescent thallium reporter dye named flu in two.
Next, the unincorporated dye is washed from the cells and replaced with assay buffer containing a vehicle control or test compounds. The final step is the addition of a stimulus buffer to the wells to initiate thallium flux. Ultimately, a fluorescence plate reader is used to identify the compounds that change the movement of thallium ions through the KIR channel.Poor.
The main advantage of this method over existing techniques such as manual patch clamp electrophysiology is that it enables the investigator to perform hundreds of experiments simultaneously and thousands of experiments daily. This method allows the Investigator to screen libraries of thousands of small molecules to discover modulators of inward rectifying potassium channels A day before the thallium flux experiment dissociate the cells and quantitate the density of the cell suspension as described in the accompanying manuscript. Next, use the BH medium containing 10%dialyzed FBS plate 20, 000 monoclonal cells stably transfected with a KIR channel gene of interest in each well of a BD pure coat Amin coated 384 well plate using a thermo multi drop combi or a multi-channel pipetter the next day, inspect the plate under a microscope to ensure that the cells are adherent and evenly distributed across the bottom of the wells.
Then use an ELX 4 0 5 microplate washer to replace the cell culture medium with HBSS assay. Buffer at 20 microliters per well. After that, prepare the flus in 2:00 AM D loading solution by briefly centrifuging the tube to dissolve 50 micrograms of flus in two powder in 100 microliters of anhydrous DMSO.
Now add 50 microliters of a 20%weight per volume onic F1 27 DMSO solution to the dye. Mix them by pipetting gently. Then add 150 microliters of flus in 2:00 AM onic F1 27 to 35 milliliters of HBSS assay, buffer and mix gently.
Next, add 20 microliters of dye loading buffer to each well that already contains 20 microliters of HBSS assay.Buffer. Incubate the cells at room temperature for approximately one hour while the cells are loading with dye. Prepare a thallium stimulus plate by freshly dissolving 0.5 grams of sodium bicarbonate in 50 milliliters of five x thallium stimulus buffer.
Then add 50 microliters of the solution to each well of a polypropylene 384 well plate After the cells have been loaded with flus in 2:00 AM.Wash the plate with an ELX 4 0 5 microplate washer. Next, add 20 microliters or 40 microliters of HBSS assay. Buffer back to each well and the plate is now ready for the experiments.
Load the thallium and cell plates into a Hema Matsu functional drug screening system or equivalent kinetic imaging plate reader with integrated liquid dispensing capabilities and use appropriate filters for fluorescein based dyes such as flu oh four. After that, set up a single add protocol to add 10 microliters of five X Thallium series to the corresponding wells of the cell plate containing 40 microliters of HBSS assay buffer. Then record the baseline fluorescence at a one hertz sampling frequency for at least 10 seconds.
The well to well fluorescence should be uniform and stable across the plate once optimal cell plating, washing and d loading conditions are established. In the meantime, use the integrated 384 channel pipetter to add the thallium stimulus buffer to each well simultaneously and continue the recording for at least two minutes so that the rate and peak of the thallium induced increase in fluorescence are captured for offline analysis. In the next series of experiments, evaluate the uniformity and reproducibility of the assay by plating a control inhibitor in every other well of each column and row of a 384 well plate and add DMSO to the other wells as a vehicle control.
Use a multichannel pipetter. Make compound DMSO plate by pipetting an inhibitor of the target KIR channel at the concentration determined previously at 80 microliters per well and 0.1%DMSO vehicle into a 384 well polypropylene plate. This is done by adding a known inhibitor starting in well A one and alternate to well B two and so on up to well B 24.
Repeat this procedure with DMSO starting in well, B one and so on up to well A 24. Now load the cell the compound and thallium stimulus plates into the plate reader. Then set up a two add protocol to add 20 microliters of the two X concentration compound series to the corresponding wells of the cell plate containing 20 microliters of HBSS assay buffer.
Add the compounds to the cell plate and incubate them for up to 20 minutes at room temperature. Record the baseline flus in two fluorescence for at least 10 seconds. Then add 10 microliters of five x thallium buffer to each well of the cell plate.
Repeat the experiment on three subsequent days to establish the reproducibility of the results. Use thallium flux in DMSO and drug treated cells to calculate a value for Z prime. A statistical measure of well to well variability among the two populations of wells calculate Z prime using this formula based on the means and standard deviations of P and N where P is the uninhibited flux value and N is the fully inhibited flux value.
An assay with ZPrime values greater than or equal to 0.5 on three separate days is considered suitable for high throughput screening. These figures are some examples of the cell plating maps used in different types of experiments. The positions of wells containing un induced or tetracycline induced cells are indicated in different colors.
This figure shows the source plate map used to determine the optimal thallium concentration for the assay development and compound screening. The color gradient represents the threefold dilution series ranging from 100%to 0.002%thallium, and here is a representative fluorescence intensity map with cool to hot colors, indicating low to high thallium flux values. The cell plating map shown here with a fit of four parameter logistic function to the thallium CRC is used to determine an EEC 80 value of 15%thallium.
This figure shows the source plate map used to determine the DMSO tolerance of an assay. Columns one and 24 contain assay buffer only, whereas the color gradient indicates the twofold dilution series ranging from 10%to 0.01%DMSO and here is a representative fluorescence intensity map with low thallium flux values indicated with darker blue. The average thallium flux values recorded from the wells containing the indicated concentrations of DMSO are summarized here.
Now this figure shows the source plate map used to establish concentration response curves for the known inhibitors of a KIR channel. Each compound is indicated with a different color and is typically plated as a threefold dilution series. A representative fluorescence intensity map is shown here, and this figure shows the dose dependent inhibition of thallium flux by-in inhibitor After its development and optimization.
The thallium flux assay can also be used to perform a variety of routine pharmacology assays. The thallium flux assay has been previously adapted for use with voltage and ligated potassium channels, non-selective cation channels, and even potassium transporters. Do not forget that working with tellium can be extremely dangerous and precaution, such as use of gloves and appropriate waste disposal should always be taken while performing this procedure.
