How to Use BioTek's Cell Count & Viability App
Hello and welcome to BioTek’s Cell Count and Viability App. This application is designed to automate hemocytometer based counting of suspended mammalian cells using BioTek imagers. The Cell Count and Viability App requires the use of phase contrast imaging. Before using the app ensure that your instrument is turned on and configured with the 4x phase contrast objective. If you need help determining if your instrument is properly configured, please contact BioTek's Technical Assistance Center.
When the Cell Count and Viability App is launched, a new blank template is generated. To navigate to previously generated data or to import previous settings, click on the menu icon in the upper left corner. Otherwise use the menus on the left to count new batch of cells.
First, click here to choose the counting slide type that you'll be using. The app is compatible with either disposable or reusable counting slides. It's important to remember that the counting slides must be loaded into the correct BioTek holder. Hover over the slide option to double check the number listed in the tool tip matches the number engraved on the holder.
For this tutorial, we'll select the disposable option. Because each disposable counting slide has two sample chambers and up to four slides can be loaded into the slide holder, you can run as many as eight samples at a time. Next, if you're using Trypan blue with your samples, ensure that the viability detection option is turned on. When the setting is on the app detects and automatically calculates the percentage of viable and non-viable cells within your samples. Because a one-to-one dilution of cell mixture to Trypan blue is assumed turning viability detection on will also set the dilution factor displayed here to 2x. The dilution factor is applied to the results so that the cell concentrations displayed later will reflect the concentration prior to the dilution and Trypan blue. If you diluted your samples further prior to loading into the counting slides, custom dilution factor can be entered. If you forget to enter a custom value initially, it can be modified anytime after image capture.
Finally, select the corresponding locations of the counting chambers that contain sample. Click on each chamber location to activate. If you'd like to provide unique names for each of your samples, enter them in the activated text boxes.
If everything looks good, check that the slide adapter is properly loaded into the instrument and then click Start.
Now that image capture is complete. Let's review the results. An image is displayed for each sample along with its calculated cell concentrations. Because viability detection was turned on, the concentrations of both live and dead cells along with their percentages are reported for each sample. Remember these results account for the dilution factor indicated here and can be modified if required.
Only results from two counting slides can be viewed at once. Click here to view the results from the other slides. You can zoom in or out of any image by using the mouse wheel. Use these buttons at the top of the screen to return all images to the same zoom level. Additionally, you can double click on any image to view it at full screen. Double click again to return to the side by side view.
The default analysis settings typically work well to accurately identify live and dead cells in your sample. However, some samples may require minor adjustments of the analysis parameters to increase count accuracy. If adjustments are needed, click the adjust analysis/image tab to access these settings. To assist with this process, it can be helpful to zoom in on a single image. Independent analyses are run for identification of live and dead cells. Live cells identified by the analysis are outlined in green, while dead cells are outlined in red. Click here to switch between highlight options. For both the live and dead cell analyses you can set the threshold and size range of cells to include. The dead cell analysis also allows you to set a limit on the roundness or circularity of cells identified as Trypan blue positive.
For quick tips on using these settings, hover over each of their labels. Whenever you adjust a setting. Click the preview button to test how your changes affect the sales identified. Click Apply to All to lock in the settings and apply them to all samples or click Cancel to revert back to the previous settings. Once the analysis is complete using your modified settings, the recalculated results will be displayed for each sample.
To export data from all samples to a spreadsheet, click the export button at the bottom left of the screen and then choose a folder location. Additionally, you can use the image export menu to export images. By default, all images are selected. Click on the individual chambers to de-select and exclude them from the export.
The Cell Count and Viability App also includes a convenient tool to assist with downstream seeding of cells at a desired concentration. Click the Dilution Calculator tab and enter values of your desired cell concentration and the final volume. If you prefer, you can enter a final cell number instead of using a concentration by selecting it in this drop down menu. After you enter your desired values, click here to run the calculator. For each sample, the calculator provides the volumes of both cell suspension, and fresh media to mix. If you'd like to modify the desired concentrations or final volumes per sample, you can change those values here in any sample box. Click the Export button to export all of the dilution calculators results to a text file for printing.
Thank you for watching this introduction to the Cell Count and Viability App. If you have any questions about this app or any other BioTek product, please contact our Technical Assistance Center using the information listed.