To begin, start the live cell analysis system software. Press the plus symbol at the top left corner of the screen to initiate the add vessel. Choose Scan on Schedule, then select New to run the scanning program.
Select Standard. Then set scan settings as cell-by-cell options to None and image channels to Phase, Green, and Red, with respective acquisition times. Set objective to 20x.
From the list, choose the plate and select the vessel location to put the plate on the tray of the imaging system. Select the wells containing samples and decide the number of images to capture per well. This information automatically generates an estimated scan duration for the plate.
Click Create Plate Map to input information for each well, such as cell type and compound. Then name the study. On the next screen, select Basic Analyzer as the analysis type and choose Analysis Definition from the dropdown list, which shows previously used analysis definitions.
Leave spectral unmixing for both green and red at 0.0. Schedule the scan to occur every 15 to 20 minutes for eight hours. Drag the white and gray bar on top of the screen to set the starting time of the scan.
On the next screen, review the scan settings, and press Add to Schedule to start scanning. After scanning, on the View tab, open the vessel for analysis. Press Launch Analysis on the left of the screen and select Create New Analysis Definition.
Choose Basic Analyzer and use Phase, Green, Red, and Overlap image channels. Select six to eight representative sample images for training. To configure the analysis definition, set green objects to neutrophils forming neutrophil extracellular traps or NETs, and red objects to the nuclei of all neutrophils.
Press Preview Current or Preview All and adjust each parameter to optimize the results. Choose the scan times and wells for analysis. Then provide a label for the analysis definition.
Review the summary and launch the analysis. After analysis, double click on the name of the analysis definition to open the analyzed study. Open Layers on the left of the screen and check if each cell is correctly marked.
Click Graph Metrics on the left of the screen, then select count per image for green count. Choose the time points and wells to be exported. For select grouping, select Plate Map Replicates to confirm all wells are correctly specified during scanning, and click on Export Data.
Similarly, export the data for red count and overlap count. Using the given equation, calculate the percentage of NETs forming cells for each condition and time point. The time course of the NET formation is visualized by plotting the percentage of NET-forming neutrophils at each time point.
Adding an AKT inhibitor blocked the NET formation in neutrophils stimulated with PMA or calcium ionophore. Demonstrating the potential molecules targeting NET formation may be tested in a high throughput manner using this methodology.
