Begin by opening the program and initialize the injector pump by clicking on the Dispenser Control, and select Pump 2, and then clicking on Init. To rinse the tubing with the ionized water, click on Rinse, then press Next. Select Pump 2 and set the tip mount to the desired tip.
Then click on Next again. Click Start to begin tube rinsing. Repeat rinsing with methanol.
Meanwhile, prepare a working substrate solution by diluting the substrate stock by 1 to 100. Finally, rinse the tubing with the working dilution of the substrate stock in PBS. Load the plate into the plate reader.
And then, on Measurement settings, choose the aperture to 384 plate US luminescence aperture, followed by the distance between plate and detector to 0.6 millimeters, and the measurement time as 0.5 seconds. Once the measurement settings have been settled, on the Protocol-General settings tab, select the plate type as 384 Nunc, and choose the By columns bi-directional option as the measurement mode. Input 1 into the number of assay repeats tab.
Now, on the Dispense tab, select the desired dispense measurement. Then set the measurement time for each well as one second and select the desired used pump. Dispensing speed of 200 microliters per second.
And the dispensing volume to 50 microliters. Choose Dispensing volume as the syringe filling volume for Pump 2. And then set the Start dispensing at measurement number to 1.
On the Well selection tab, use the Add/remove samples dropdown menu and choose the desired sample type, and select the wells to be measured. After that, start the measurement by clicking Run. The basal luminescence signal from DMSO is seen in column 1, and the decrease of luminescence signal on the presence of ATG4B inhibitor, FMK-9a, is seen in column 24.
