This method can help answer key questions in chemical biology and drug discovery. Such as, does your compound bind to the target protein? The main advantages for this technique are that there's no requirement of detachment of the adherent cells.
And spatial localization can be determined by imaging. Though we've applied this method in cell lines, it's also possible to apply this method to other cell systems such as primary cultures and cocultures. Before seeding the cells, place black 384-well imaging assay plates in a tissue culture hood, and cover the plates with aluminum foil before using a standard drill to make three 3.5-millimeter-diameter holes on both ends of each plate.
When the plates are ready, use aseptic technique to wash an A-431 cell culture with 5 to 10 milliliters of PBS. And incubate the cells with two milliliters of trypsin at 37 degrees Celsius until the cells detach. After counting, dilute the cells to 5 times 10 to the 4th cells per milliliter in culture medium, and seed 40 microliters of cells to each well of each assay plate.
Gently shaking each plate from side to side after seeding to ensure a homogenous distribution of the cells across the well bottoms. To minimize plate edge effects, allow the cells to settle at the bottom of the assay plates for 20 minutes at room temperature in the back of the laminar flow hood before placing the plates in a custom humidified chamber for two to three days at 37 degrees Celsius and 5%carbon dioxide. On the day of the experiment, use a plate washer to aspirate the medium from each well.
And add 30 microliters of the compound of interest at the appropriate experimental concentration to the appropriate experimental wells. Seal the compound-treated assay plates with breathable plate seals. And return the plates to the cell culture incubator for 30 minutes.
To expose the cells to a heat challenge, first set the water bath to the appropriate experimental temperature, and place an unsealed dummy plate containing the same volume of medium per well as the assay plate into the bath, along with a thermocouple thermometer for monitoring the temperature within the wells. When the appropriate experimental temperature has been reached, remove the breathable seal from each assay plate and reseal the plates with a tight adhesive aluminum foil to ensure that no water leaks into the wells during their subsequent heating in the water bath. Keeping the drilled holes within the plate frame accessible.
Place the assay plates and a new dummy plate into the water bath, with the bottoms of the plates angled toward the water surface to force any remaining air from under the plates, and begin monitoring the temperature of the new dummy plate. An even heating of the plate is critical for assay performance. Take care to place the plate in the water bath such that there are no air bubbles trapped underneath.
After three minutes, immediately cool the plates in a second water bath of room-temperature water for five minutes before their analysis. To image the cells, first add 10 microliters of 16%paraformaldehyde directly to the assay plates for a 20-minute incubation at room temperature. At the end of the fixation period, wash the cells with 300 microliters of PBS on the plate washer, and add 20 microliters of 0.1%NP40 for a 10-minute incubation at room temperature.
At the end of the incubation, wash the cells as demonstrated. And block the cells with 15 microliters of 1%bovine serum albumin, or BSA, for one hour at room temperature. Next, use the plate washer to aspirate the blocking serum, and add 10 microliters of the primary antibody of interest to the appropriate wells for a one-hour incubation at room temperature.
At the end of the incubation, wash each well with 300 microliters of PBS, and label the cells with 10 microliters of the appropriate secondary antibody for one hour at room temperature protected from light. For nuclear staining of the cells, add 10 microliters of an appropriate nuclear dye to each well for 10 minutes at room temperature. And wash the cells in PBS as demonstrated.
Label the cells with 10 microliters of cell mask per well for 30 minutes at room temperature. Followed by a last wash with PBS. Then dispense 60 microliters of fresh PBS to each well, and seal the plates with aluminum foil until imaging.
To image the cells, capture four images per well on a high-content imager using the appropriate fluorescent channels under the 10-times objective using automated laser autofocus and apply binning to during the acquisition. Then store the images as 16-bit grayscale dot-tiff files along with the metadata. Antibody recognition should not be disrupted by conformational changes in the target protein that may be induced by ligand binding.
For example, BIRB796 has a long off rate, and quantification of the target engagement is only possible by applying an antigen retrieval step. This representative thermal aggregation curve experiment for cells treated with positive and negative control compounds illustrates typical protein stabilization ranges after treatment of the cells with the compounds at various temperatures. Here a typical isothermal dose-response fingerprint experiment is shown to demonstrate representative ranges of protein stabilization in response to different doses of experimental compound.
The application of isothermal heat challenges for compound screening to identify novel binders of the target protein allows the analysis of a large number of compounds at a single concentration at one time, before isothermal dose-response fingerprinting of the hits to confirm target protein stabilization. While attempting this procedure, it's important to remember that the exact experimental conditions, such as the length and temperature of the heat challenge impact the observed potency of the compounds. Don't forget that working with paraformaldehyde can be extremely hazardous.
And precautions, such as following institutional safety guidelines, should always be taken while performing this procedure.
