Rainbow Linear Calibration Particles

The use of flow cytometry to analyze small particles has been practiced for several decades. More recently, due to the increased sensitivity of conventional and commercially available flow cytometers, along with the growing interest in other small particles, such as e.g. B. extracellular vesicles, increasingly used. Despite an increasing use of small particle flow cytometry, the lack of standardization in data reporting has resulted in a growing body of literature on extracellular vesicles (EVs) that cannot be easily interpreted, validated, or reproduced. Methods for standardizing fluorescence and light scattering are well established and the reagents for performing these analyzes are commercially available. 

Here we outline FCM  _PASS as a software package to perform fluorescence and light scattering calibration of small particles while generating standard reports that conform to the MIFlowCyt-EV standard reporting framework. This unit covers the workflow of performing calibration with FCM  _PASS  as follows: Acquisition of fluorescence and light scattering calibration materials, cataloging of the reference materials for use in the software, creation of cytometer databases and datasets to link calibration data and fcs files,Import fcs files for calibration, enter fluorescence calibration parameters, enter light scattering calibration parameters and apply calibration to fcs files.

INTRODUCTION:

The FCM  _PASS  software is designed to perform calibrations and keep records on multiple instrument platforms. The workflow is designed to be ergonomic for core flow cytometry facilities and researchers with multiple instruments interested in longitudinally tracking their instrument performance and maintaining records of their experimental calibration.

•  To enable this, the FCM  _PASS  software focuses on maintaining databases for each cytometer, adding a record for each calibration (  Protocol  ).
•  For ergonomic use and to maintain high-quality report results for publication, the FCM  _PASS uses a reference materials cataloging system (  Protocol ). 
• The reference material catalog allows users to enter complete records of their calibration reference materials only once.
•  When this information is stored, it can be used as a reference when performing future calibrations and allows full documentation in the automated calibration reports.

FCM  _PASS  is designed to perform fluorescence calibration for any number of fluorescence parameters with the ability for the user to

to change the method of unit scaling for regression, along with defining fluorophore-to-protein ratios to convert outputs from molecules of equivalent soluble fluorophores (MESF) to epitope number (Protocol 7). This unit covers acquisition and control of fluorescent calibration materials for sample data calibration (  Protocol 2  ) or cross-calibration of other fluorescent reference materials (  Alternate Protocol 2  ).

Light scattering calibration using FCM  _PASS  software is a semi-automated process, with the software approximating the collection half-angle to generate a scattering diameter curve (  Protocol 8  ). This unit covers the acquisition and control of light scattering calibration materials for sample data calibration (  Protocol 1  ). By default, the software outputs core-shell models based on light scattering properties of extracellular vesicles. However, the software allows users to customize core-shell model outputs along with homogeneous sphere outputs.

Finally, once the calibration is done, all the calibrated parameters are written to .fcs files that preserve the metadata from the source file (  log 9  ). This allows users to share their data in calibrated units. After calibration, quality control plots for light scatter and fluorescence are output along with a table. The table contains a MIFlowCyt-EV report with the calibration fields automatically filled in by the software (  Welsh, Van Der Pol, et al., 2020 ). This table also includes the detection limits for files that used a trigger threshold with a calibrated parameter. All metadata associated with fluorescence and light scattering calibration to enable reproducibility is also shared.