The overall goal of this procedure is to develop a single day cell-based cyclic A MP sensitization assay for high throughput screening applications, including chemical library screening. This is accomplished by first thawing and plating previously prepared assay ready cryopreserved cells. The second step is to add test compounds or antagonists to the cells, followed by the addition of the adenyl cyclase sensitizing drug.
Next adenylyl cyclase activator is added in the presence of phosphodiesterase inhibitor to stimulate cyclic A MP accumulation. The final step is to quench the reaction with homogenous time resolved fluorescence or HTRF cyclic A MP detection, reagents in lysis buffer, and then measure fluorescence. Ultimately the use of cryopreserved cells and HTRF cyclic A MP technology allows for the execution of a single day sensitization assay for small molecule screening.
We first had the idea for this approach when we became interested in using high-throughput screening to study receptor mediated heterologous sensitization of a dental cyclase. Two key techniques used here include using cryopreserved cells as off the shelf reagents and homogeneous time result. For essence assay kits for measuring cycle KMP, The basic methodologies used here can help answer key questions in cell signaling and drug discovery using cell-based assays To begin culture CHO D two L cells on a 15 centimeter cell culture dish as described in the text protocol, incubate the cells at 37 degrees Celsius and a humidified incubator with 5%CO2 until the cells are 90 to 95%Confluent then wash the cells with 10 milliliters of phosphate buffered saline or PBS harvest the cells and three milliliters of cell dissociation buffer following a five minute incubation at 37 degrees Celsius.
After Resus suspending the cells using 12 milliliters of the culture media, count the cells using trian blue exclusion centrifuge the cell suspension at 500 Gs for five minutes at room temperature. Then aspirate the sane and resus, suspend the cell pellet in five milliliters of freezing media after diluting the cell suspension to achieve the desired cell concentration, aliquot one milliliter of cell solution to each cryo vial. Incubate the cryo vials in a cell freezing container at negative 80 degrees Celsius overnight.
The following day, transfer the cryo vials to a liquid nitrogen tank for long-term storage to plate assay ready cells first rapidly thaw a frozen cryo vial of cells and a 37 degree Celsius water bath. Once cells are thawed, transfer the cells to a 15 milliliter conical tube containing nine milliliters of optimum and mixed by inverting the tube three to five times following centrifugation of the cells at 500 Gs for five minutes. At room temperature, aspirate the supernat and resuspend the cells in one milliliter of optimum.
Count the cells using trian blue exclusion to determine cell viability before diluting the viable cells as necessary. And optimum plate 10 microliters of cells per well and a tissue culture treated 384 well plate. Using a multichannel pipette.
Make serial dilutions of cyclic A MP and optimum to generate a standard curve for estimating cyclic A MP production by the cells. Add 10 microliters per well of the cyclic A MP standards to the plate. Centrifuge the plate at 100 GS for 15 seconds at room temperature and incubate at 37 degrees Celsius and a humidified incubator with 5%CO2 for one hour.
For a small molecule screening, first dilute the drug of interest in optimum to six times the desired final concentrations. Serial dilution can be completed using handheld pipettes or a liquid handling station. Add 2.5 microliters per well of the test, compound or buffer containing vehicle.
Apply the solution to the side of the wells using a multichannel pipette centrifuge the plate at 100 GS for 15 seconds. At room temperature for persistent agonist treatment, prepare a 600 nanomolar solution of Quin parol and optimum then at 2.5 microliters per well of the 600 nano molar Quin parol solution to the side of the wells. Using a multi-channel pipette centrifuge the plate again before incubating at 37 degrees Celsius and a humidified incubator with 5%CO2 for two hours.
During the two hour incubation, prepare the stimulation solution in optimum as described in the text protocol. Add five microliters per well of the stimulation solution to the side of the wells using a multichannel pipette after centrifuging the plate as before incubated room temperature for one hour. Production of cyclic a MP by the cells is measured using the cyclic A MP dynamic two kit according to manufacturers instructions briefly, reconstitute anti-cyclical A PK crypt and cyclic A PD two in distilled water dilute one aliquot of anti-cyclical A MPK crypt and one aliquot cyclic A MPD two separately in the lysis buffer, according to the manufacturer's instructions to make working solutions to quench cyclic a MP accumulation in the 384 well plate add 10 microliters per well of the anti-cyclical A MP crypt eight working solution and 10 microliters per well of the cyclic A MPD two working solution.
Using a multichannel pipette centrifuge the plate again at 100 GS for 15 seconds at room temperature before incubating at room temperature for one hour, read the plates in a fluorescence plate reader using an excitation of 337 nanometers and measure the emissions at 620 nanometers and 665 nanometers per the manufacturer's instructions. Apply ratio metric analysis to assess the cyclic A MP standard curve per the manufacturer's instructions. Using the resulting values extrapolate the estimated cyclic A MP accumulation in the test wells using data analysis software.
Cryopreserved CHO D two L cells were plated directly into 384 well assay plates to demonstrate that sensitization assays can be performed in a 384 well format reducing the number of steps from more than 20 to five. The studies revealed that a two hour pre-treatment with Quin Perol enhanced subsequent four stimulated cyclic A MP accumulation in a dose dependent manner consistent with heterologous sensitization. The second series of experiments explored whether this new streamlined assay could be utilized to assess inhibitors of sensitization.
The initial results revealed that pre-treatment with haloperidol orpi Perone prototypical D two antagonists completely prevented Quin parol induced sensitization of four stimulated cyclic A MP accumulation. This method was then used to assess the potency of a series of D two antagonists to test whether these compounds inhibit sensitization similar to the previous results. These studies demonstrated that the D two antagonists inhibited agonist induced sensitization in a dose dependent manner.
The next objective was to develop a 384 well sensitization assay that can be used to conduct scalable reverse transfection SI RNA library screening as described in the text protocol, the results revealed that exposure of cells to the agonist Quin parole for two hours led to a marked enhancement of four skull and stimulated cyclic A MP accumulation. The increased accumulation of cyclic A MP at both 100 nanomolar and 300 nanomolar of four scullin was greater than 15 fold as compared to vehicle treated cells. Reverse transfection of GSS irna into D two receptor expressing cells blocks agonist induced sensitization of adenyl cyclase.
When the D two receptor agonist Quin Perol was added and cyclic, a MP accumulation was stimulated and measured using H-T-R-F-A 15 fold sensitization response was reduced by 94%Once mastered, this entire assay can be done in less than eight hours if it's performed properly Following this procedure. Other signaling molecules like IP one and Phospho Earth can also be measured in order to examine additional receptor signaling pathways.
