Acquisition of images to monitor the effect of acetate (activation test) and the compound tested (high-throughtput screening) on the calcium activity of the cells over time :
Implementation of image acquisition :
For each well, three images are first acquired in the absence of the compound (in the case of activation test : the initial 20mM acetate and for the screening experiment : the compound to test) and the effect of the latter on the cells is followed after the addition of the compound from the 4th to the 12th image.
Equipment and reagents required:
HCS microscope
The cell plate in the robotic incubator.
The untreated 96-well plate (ref. 3370) identified. This is an activation plate.
2 racks of cones "ref 9000-0761; 96-Well, FLIPR Tetra Pipet Tips (Clear)" adapted to the fluidics module of the HCS microscope.
Launch the MetaXpress software by double-clicking on the "MX" shortcut.
Open the image acquisition protocol by clicking on "Acquisition Setup" in the "Screening" menu of the software.
Open the trap door of the acquisition chamber by clicking on "Eject Plate"
Place the cell plate in the acquisition chamber with the A1 side to the left (towards the objectives)
Remove the lid from the cell plate to avoid disturbing the addition of compounds from the compound plate.
Close the acquisition chamber by clicking on "Load Plate".
Load the image acquisition protocol by clicking on "Load Protocol" then "Load From Plate”
Load the dedicated Hutu-80 or NCI h716 GCaMP cell acquisition protocol
Select the wells to be imaged by the microscope. They should appear in green.
Select the image acquisition wavelength by clicking on "Active Wavelength" and check that the exposure time (Exposure) is as specified in the acquisition protocol.
If the sharpness (or focus) of the image is not satisfactory, click on "Calculate Offset".
Using the image scroll button, select the sharpest image and click on "OK".
In the "Run" tab, give a name to the folder (Folder Name) that will contain the plate images.
Give a name to the plate (Plate Name) whose images will be taken by the microscope.
NB: These names should be short in order to facilitate their registration in the database. If you wish to add information that you consider important, you can note it in the "Description" box.
It will not be necessary to save the acquisition protocol, as it already exists.
Remove the cover from the compound plate.
Click on "Acquire Plate" to start image acquisition.
From one of the PCs with the MX software installed, extract the raw image segmentation data.
This data will be extracted into an Excel file.
Analyse the data.
By performing the ratio of the average fluorescence intensities before and after addition of the compound.
Before analysing the data, check that the activation of the cells by the acetate is in accordance with the expected results (see analysis of activation test results). If this is not the case, report the non-conformity and try to find the cause. Depending on the case, refer to the process pilot to know if the analysis should be carried out or not. All screening analysis files should be saved on the relevant folder. Analyse the data the same way as the activation test.