This protocol offers a quick and efficient strategy to simultaneously use two assays to screen several hundred compounds for their effects on cell growth. The assays can provide us information on whether specific compounds are cytotoxic, inhibit cell growth, or enhance cell growth. By utilizing the two assays together, we are able to increase accuracy and identify cytotoxic compounds.
In cases where the results of the two assays diverge, this strategy may allow us to identify compound with unique effects on cellular function that can be further examined by other means. The main advantage of this strategy is that it is quick, inexpensive, and non-labor intensive. Once mastered, it allows researchers to easily perform a primary screen of up to 200 compounds in one week, and then perform an exhaustive dose response curve on 10 to 15 compounds in another week.
Demonstrating the procedure will be myself and Rohini Manickam, a staff scientist and post-baccalaureate fellow from the laboratory. First, in a biological safety cabinet, culture cells in a T75 flask with NSC medium so that they will be at least 80%confluent when beginning the assay. And place the flask in a cell culture incubator set at 37 degrees Celsius and five percent carbon dioxide.
Once the cells reach 80%confluence, remove the flask from the incubator and aspirate off NSC medium. Add three milliliters of cell dissociation reagent and incubate for five minutes in the incubator. After incubation, add seven milliliters of NSC medium in the flask, and pipette vigorously to ensure all cells become detached.
Transfer the dissociated cell solution to a 15 milliliter tube, and centrifuge at 200 times G for five minutes. Remove the supernatant from the tube and re-suspend the cells in 10 milliliters of NSC medium. Use an automated cell counter or hemocytometer to count the cells, and add NSC medium into the tube to readjust the concentration of cells to 200, 000 cells per milliliter.
Then, using six of the eight slots of an eight channel multichannel pipettor to plate 100 microliters of the cell mixture column by column in 60 interior wells of three 96 well plates that have been coated by extracellular matrix. Add 100 microliters of base medium or NSC medium to all wells without cells to minimize potential evaporation from outermost wells containing cells. Under a cell culture microscope, visually inspect at least 10 wells on each of the three 96 well plates to confirm that the cells are seated at the expected density.
Incubate at 37 degrees Celsius and five percent carbon dioxide. Remove the cell culture plates from the incubator 16 to 24 hours after splitting, and aspirate off NSC medium column by column with an eight channel multi-well pipettor, using only six of the eight multi-well slots. Add 95 microliters of fresh NSC medium to each well of the three replicate plates, and place the plates back into the incubator.
With an eight channel multi-well pipettor, add 49 microliters of NSC medium into each of the interior 60 wells of an empty U-bottomed, V-bottomed, or round-bottomed 96 well plate. Unseal the prepared master compound plate. Pipette one microliter of the compound from the master plate into each of the interior 60 wells containing NSC medium, and mix the diluted compound three times with a bench top pipettor.
Next, remove the three 96 well plates of NSCs from the incubator and dispense five microliters of the diluted compound into each well of the three plates that contain cells to yield a one to 1000 dilution for the final compound concentration of 10 micromolar with a DMSO concentration of 0.1%Incubate cells with compound for 72 hours, and proceed with cytotoxicity assays. To perform dose response assay, set up a 96 well. Use Column Two for six DMSO control replicates, and test triplicates of up to three different compounds at twofold serial dilutions, at six doses, starting with a high dose of 10 micromolar.
Dilute four microliters of 100%DMSO or test compound in DMSO into 196 microliters of NSC medium in a 0.5 or 1.5 milliliter microcentrifuge tube. Add 25 microliters of the diluted DMSO to control wells, and 50 microliters of the diluted test compounds to their corresponding wells. Pipette 25 microliters of NSC medium to the remaining empty wells in the interior portion of the 96 well plate.
With a multichannel pipettor, transfer 25 microliters of the compound from the 10 micromolar wells to the five micromolar wells, and mix at least five times. Replace the pipette tips, and repeat the process by mixing the remaining rows to generate triplicates at twofold dilutions for a total of six doses for each of the compounds. Transfer five microliters of each dilution into a new plate with culture cells that are being tested.
Place the plate in the incubator at 37 degrees Celsius and five percent carbon dioxide. After cells have been incubated for 72 hours, remove the plate from the incubator and place it inside the plate reader. Open the imager software to set up protocol and experiment files for the study.
Go to Imager Manual Mode on Task Manager and click Capture Now. Choose 96 well plate as the vessel type. Select 10 times for the magnification, and red fluorescent protein 531 and 593 for imaging tdTomato.
Click on a well. Then click on Autofocus to focus image, and Auto Expose for proper exposure time. Manually adjust the focus and exposure if needed.
Click the camera icon to capture the picture. Then click Process Analyze above image to continue building the protocol. And select the Analysis tab.
To the right of the image, click Cellular Analysis in Add Analysis Step, and click Start. The image shows highlighted cells to indicate each individual cell. Click on the Options selection to alter parameters to better select cells based upon the fluorescence threshold or cell size.
If the imager is properly counting the cells, then click Add Step at the bottom of the screen. Click the icon at the top of the screen to create experiment from the image set. And on the opened window, click Procedure under the Protocol tab.
Then, in the new window that opens, select Read. Click Full Plate to select only the 60 wells that contain the cells. Click OK to save changes.
Then click OK in the Procedure window. Click the Play icon to run the plate. In the prompt, save the file, and then in the Load Plate prompt, click OK.Upon completion of the imaging, download the cell count data from the imager software program to a spreadsheet for analysis.
Within two hours of completing tdTomato imaging, begin the MTT assay. Weigh out 25 milligrams of MTT and re-suspend it in five milliliters of NSC medium to make five milligrams per milliliter MTT stock solution. Vortex the solution for several minutes, until there are no visible precipitates of MTT.
Remove cell culture plates from the incubator and aspirate off cell culture medium. Dilute MTT one to 10 in fresh NSC medium, and add 100 microliters of the diluted MTT to each well of cells. Incubate cells at 37 degrees Celsius for two hours.
A purplish precipitate is visible roughly in proportion to the number of cells in the well. Aspirate the MTT solution off the plates, and add 50 microliters of 100%DMSO to each well. Place the plates on a shaker at room temperature for 10 minutes at 400 RPM.
After that, transfer the plate to a plate reader. Read the absorbance of each well at the preset wavelength of 595 nanometers, and export data to spreadsheet for analysis, exactly as for the cell count data. Examination of tdTomato fluorescence images revealed two wells discordant for toxicity between the MTT and cell count assay.
For one well, cell count data suggested no toxicity. But MTT data did. Another well had overestimated cell count by MTT relative to tdTomato count, and appeared to have larger cells, whereas in wells where MTT underestimated cell count relative to tdTomato, the cells appeared to be smaller.
In summary, the tdTomato assay classified 11 compounds as toxic, 11 as growth inhibitory, and one as growth enhancing, with 34 having no apparent effect on cell growth. The MTT assay classified 13 compounds as toxic, two as growth inhibitory, and one as growth enhancing, with 41 having no apparent effect on cell growth. The graph of viable cells for the compound WP1066 shows a relatively flat curve with no toxicity for the four lowest concentrations.
The curve falls rapidly at the five micromolar dose, and drops to nearly full toxicity at 10 micromolar, which leads to the lethal dose 50 as 4.4 and 6.0 micromolar for the tdTomato assay and the MTT assay. It is critical that compounds are properly diluted in the dose response screen so that the correct dose response curve is generated. Compounds identified by this procedure to affect cell growth can be further characterized by functional assays to identify potentially novel signaling pathways that control cell growth.
Compounds that are toxic to neural stem cells have been tested as potential therapeutic agents for brain cancers, such as glioblastoma and neuroblastoma. Always wear gloves and a lab coat when handling mammalian cells, DMSO, and MTT. Dispose DMSO and MTT according to the Chemical Waste Guidelines of your institution.
