To begin, place the sensing lid on an empty 96 well receiver plate and place it in the cell culture incubator. Align and mount the device on the sensing lid and connect it to the hub via the provided USB cable creating a Recypher sandwich. Now go to the Lucid Lab website and click the new experiment button on the top right corner to create a new experiment.
Name the title of the experiment and input any relevant experimental notes for the study. Next, create well conditions in treatment groups. For example, if testing the effects of different serum concentrations in the media on oxygen consumption rate, select serum in group name intermediate serum concentrations to be used, and add more serum percent values by clicking the plus button.
Then assign which wells will receive a particular serum percentage and select a color pattern for those wells. Click the add group button to add more groups as needed. Next, define the plate setup and select the corresponding device and plate style.
To do so, click the add plate button, choose the device and select the plate type. Select treatment, and click on the corresponding well. Now, click the start now button to start the experiment.
Check that the indicator on the website and the light emitting diode on the hub is solid green. Let the system run for 15 to 60 minutes to allow for testing that each sensor is well calibrated and detecting atmospheric oxygen. Afterward, remove the device from the sensing lid and place the device upside down in the incubator to allow the motor on the device to reset.
Now, place the plate with the sensing lid back in the cell culture hood, along with a 96 well plate containing the RPE cultures. Change the media in the plate with RPE cultures to the same media and volume for all wells to obtain baseline oxygen consumption rate values for each well. Then transfer the sensing lid to the plate containing cell cultures and place the standard lid on the plate containing cell cultures to the empty 96 well receiver plate, to maintain the sterility of the standard lid.
Place the plate with cell cultures and the sensing lid back in the cell culture incubator. Align and mount the device on the sensing lid and connect it to the hub via the provided USB cable. Check that the indicators on the website interface and the hub are all solid green.
Let the device measure oxygen consumption rates or OCR on each well for at least 12 to 24 hours to capture a baseline OCR. After the experiment, immerse the sensing lid in 70%ethanol for 10 minutes in a cell culture hood to sterilize it. Once the ethanol and water totally evaporate, put the sensing lid on a new 96 well receiver plate.
