The standardization method of the flow cytometer provide a feasible approach for the mutual accreditation of laboratory results and ensures the consistency of results with research projects across multiple centers. The method minimize time consumption is easy to execute, is equipped to automate, analyzes template, and can dramatically reduce the requirement of data analysis. Begin creating a new configuration in the software of cytometer A, from the transferring lab, by clicking the cytometer button, and choosing view configurations.
Right click the base configurations folder in the configuration list, choose new folder and rename it. Next, create a new configuration by right clicking the base configuration and copying it. Right click the new folder and paste the base configuration before renaming the new configuration, Standardized method transfer"Drag the parameter name, such as, FETC from the parameters list, onto the appropriate detector 530/30, also edit additional parameters to the new configuration.
To standardize the experiment in the transferring lab, run a performance check using this cytometer A setup and track the beads to verify that the cytometer A is performing well. Right click cytometer A settings in the software browser window and create a worksheet by selecting the application settings. Then use an unstained sample to adjust the photo multiplier tube, or PMT voltages, of the forward scattering area, or FSC, and side scattering area, or SSC, and all fluorescence parameters.
Save the application settings by right-clicking the experiment cytometer settings and selecting application settings. To add compensation controls automatically, click on the experiment button and select the compensation setup menu, before selecting create compensation controls. Record data for all compensation control beads.
Once done, click on the experiment and select compensation setup, calculate the compensation automatically, before recording the data for single cell stained controls. use CD45 RAA APC to modify the compensation value of R7-18 to 15%APC, use CD19 BB700 to modify the compensation value of APC H7 to 14%BB700, and use CD8 R7-18 to modify the compensation value of APC H7 to 60%R7-18. After recording the data for the CST beads, create a global worksheet of the target value template for the CST bright beads, once done, run the samples on the flow cytometer array and collect 25, 000 lymphocytes.
Create a global worksheet of the analysis template for the samples and save the experimental template on cytometer A.Export the experimental template using the CD-ROM. To transfer the experimental template to cytometer B in the test lab, conduct a performance check using CST beads to verify that cytometer B is performing well. Import the experimental template from cytometer A and create an experiment for cytometer B using the template.
Using the same lot of CST beads adjust the fluorescent parameter voltages for each fluorescence channel to match the previous MFI instrument. If needed, adjust the FSC voltage using the unstained sample. Right click on the experimental cytometer settings and select application settings to save the application settings.
Next, modify the compensation value of R7-18 to 15%APC using CD45 RA APC. Use CD19 BB700 to modify the compensation value of APC H7 to 24%BB700. And use CD8 R7-18 to modify the compensation value of APC H7 to 60%R7-18.
After compensation value modification, run the samples on the flow cytometer B and collect 25, 000 lymphocytes. In a global worksheet of the target value template for the cytometer setup and tracking, or CST bright beads, the histogram plots of 10 fluorescence channels were obtained, the target value for each parameter was displayed by showing the median within the histogram gates. the dot plots of the sample obtained using the cytometer A and B after instrument standardization, demonstrated the consistency of data between different instruments using an automatic analysis template.
No significant difference was observed when the results from six samples were compared across two different instrument models. The results of 15 lymphoid subsets obtained from different instruments using the standardized method acquired highly comparable data. The same configuration name, creating the template, and making the ecotech values of safety beads in different instruments are critical steps in this protocol.
