To begin, take a tube with patient-derived organoids or PDO suspension in an equal amount of basement membrane extracts, or BME. Using a 20 microliter pipette, seed five microliter domes into the center of each well of a pre-warmed 96 well plate. After seeding all the wells, place the lid on the plate and gently invert it.
Incubate the inverted plate at 37 degrees Celsius for 20 minutes in the tissue culture incubator to allow the domes to solidify. Flip the plate over so that the lid is facing upwards. Then incubate again.
After adding fluorescent dyes and drugs to the plates containing the PDOs. Place the plate in citation five. Start the Gen5 software and in the task manager window, select imager manual mode and click capture now.
In the settings, specify the objective, filter, microplate format, and vessel type, select use lid and use slower carrier speed and click okay. Then choose a well of interest. Select the brightfield channel, click on auto expose and adjust the settings as required.
Next to set up the Z stack, expand the imaging mode tab, select Z stack, and adjust the focus using the course adjustment arrows until all PDOs are out of focus. Establish this point as the bottom of the Z stack and repeat in reverse to set the top. Afterward, set up the fluorescent channels by opening the edit imaging step.
Under channels, select the desired number of channels, designate one channel for the brightfield and additional channels for each fluorescent channel. Close the window by clicking okay, change the dropdown menu to the GFP to set the exposure for the first fluorescent channel, click auto expose and adjust the exposure settings under the exposure tab to reduce the background signal. To copy exposure settings to the image mode tab, click the copy icon and then click edit imaging step to open a new window.
In the GFP channel, click the clipboard icon in the exposure line to add settings for illumination, integration time, and camera gain to the channel. Now click on the camera icon and choose image pre-processing under add processing step. On the brightfield tab, uncheck the apply image pre-processing option.
For each fluorescent channel tab, ensure apply image pre-processing is checked. Deselect, use same options as channel one, and then click okay to close the window. Now click on Z projection under the add processing step, adjust the slice range if necessary, and close the window by clicking okay.
To set up the protocol for kinetic imaging, in the toolbar click image set, and from the dropdown menu, choose create experiment from this image set. Click on options under the other heading and check the discontinuous kinetic procedure box. Under estimated total time, enter the runtime for the experiment.
Close the window by clicking okay. Additionally, set plate layout and temperature gradient at this time. Validate the data reduction steps by clicking through the windows.
Then navigate to file and click save protocol as in the toolbar. Select a save location. Enter a file name and click save.
To begin kinetic imaging, load the protocol into the bio spa on demand scheduling software and choose an available slot. In the procedure info tab, select user from the menu. Next to the protocol slot, click select and choose add a new entry.
Now click select next to the protocol slot. Click open to navigate and import the protocol into the scheduling software. Enter the required imaging time for the plate and click okay to close the window.
Under interval, input the desired frequency of imaging, select schedule, plate, or vessel to initiate the plate validation sequence, click yes to accept this schedule.
