A Cellular Assay for the Identification of CXCR4-Interacting Agents
Transcription
On the day of cell passaging, coat each well of a black-walled polystyrene 96-well plate with clear bottoms with 100 microliters of 0.1% gelatin solution, and incubate the plates for two hours at room temperature. While the gelatin is solidifying, use a trypsin-EDTA solution to lift the cells of interest from the bottom of their culture flask, resuspending the cells in fresh growth medium in a 50-milliliter conical tube once they have detached.
After counting, collect the cells by centrifugation, and resuspend the pellet in fresh growth medium to a 1 x 105 viable cells per milliliter concentration. When the plates are ready, discard any excess gelatin solution, and dry the plates on a lab tissue. Then, wash the wells with 200 microliters of PBS, and replace the wash with 200 microliters of cells per well, for an overnight incubation at 37 degrees Celsius and 5% carbon dioxide.
The next morning, tap the plate to discard the growth medium from the wells, and dry the plate upside down on a paper towel. Then, label the cells in each well with 100 microliters of loading dye solution per well, and incubate the plate for 45 minutes in the dark at room temperature. While the cells are incubating, add 75 microliters of CXCL12 per well to the chemokine plate, and 50 microliters of the compound of interest to each well of the compound plate.
Next, turn on the cooler unit of the fluorescence plate reader, followed by the plate reader itself. After allowing the system a few minutes to initiate, open the plate reader software. To set up the assay protocol, under the Settings tab, select Read Mode, and the appropriate excitation and emission wavelength for the loading dye.
Select the "Mix with Transfer Fluid" tab for automatic mixing of the compounds in the compound plate. In the "Transfer Fluid" tab, set 20 microliters of each well from the compound plate to be transferred into the measurement plate, and position the pipette tips at 20 microliters below the liquid surface. Set the speed of aspiration to 50 microliters per second, and the speed of dispensing to 25 microliters per second and click "Read With Transfer Fluid."
During the first interval, select 60 reads with a read interval time of one second with 10 reads to be recorded before the compounds are dispensed into the measurement plate, and 50 reads to be completed after. Set the second interval with the read interval time of 30 seconds and 18 reads. Select three tip washes for the protocol, setting each wash step to include the appropriate wash fluid, one wash cycle, a fast pump speed, and five strokes per wash.
Use the "Pause Pipettor" function to include a pipettor pause of 300 seconds and use the "Mix with Transfer Fluid" setting to allow automatic mixing of the CXCL12 solution within the chemokine plate. In the subsequent "Transfer Fluid" tab, set 25 microliters from each well of the chemokine plate to be transferred to the measurement plate and position the tips 20 microliters below the liquid surface. Set the aspiration speed to 50 microliters per second, and the dispensing speed to 25 microliters per second.
Use the "Read with Transfer Fluid" button to set the first interval to include 145 reads with a read interval time of one second. Set the second interval to a read interval time of six seconds and 20 reads. Select three more wash tip cycles, then click "Set Stage Temperature" and set the device temperature to 37 degrees Celsius.
At the end of the loading dye incubation, discard the buffer from the measurement plate and dry the plate on a lab tissue. Wash the cells with 150 microliters per well of assay buffer, replacing the buffer with 80 microliters of assay buffer after two minutes. Place the measurement plate in the read position, the compound plate at the source 2 position, the chemokine plate at the source 3 position, and a box of black tips at the source 1 position.
Close the device and click "Protocol Signal Test" to determine the background relative light units and fluorescence variants over the plate. Select Test "Signal" in the pop-up window. If background values of 8,000 to 10,000 relative light units are obtained, select "Update" and click "Save" to save the main protocol.
