TITLE: Resource 2:  Fluorescence Detector Setting Incrementation for FCMPASS
AUTHORS: Joshua A Welsh, Sean M Cook, Jennifer Jones

[DESCRIPTION]
Flow cytometry (FCM) is a common extracellular particles (EPs), including viruses and extracellular vesicles (EVs), characterization method. Frameworks such as MIFlowCyt-EV exist to provide reporting guidelines for metadata, controls, and
data reporting. However, tools to optimize FCM for EP analysis in a systematic and quantitative way are lacking. Here, we demonstrate a cohesive set of methods and software tools that optimize FCM settings and facilitate cross-platform comparisons for EP studies. We introduce an automated small particle optimization (SPOT) pipeline to optimize FCM fluorescence and light scatter detector settings for EP analysis and leverage quantitative FCM (qFCM) as a tool to further enable FCM optimization of fluorophore panel selection, laser power, pulse statistics, and window extensions. Finally, we demonstrate the value of qFCM to facilitate standardized cross-platform comparisons, irrespective of instrument configuration, settings, and sensitivity in a cross-platform standardization study utilizing a commercially available EV reference material.

[STEPS]
SECTION: Sample preparation
1. Vortex QbSure bottle on a high setting for 5 s. 

 

SECTION: Sample preparation
2. Pipette 500 µL of DPBS to two FACS tubes. Label one tube 'DPBS" and the second tube 'Beads'.

 

SECTION: Sample preparation
3. Add 3 drops of QbSure beads to the 'Beads' tube and vortex for 5 s. 

SECTION: Cytometer Setup
4. Ensure cytometer is clean and that -Height and -Area statistics are set to be collected on all parameters and that all parameters are on.
SECTION: Cytometer Setup
6. Create a pseudocolor plot with FSC-H on the X-Axis and (488 nm) B-SSC-H on the Y-Axis and make sure both parameters are being plotted on a linear-scale.
SECTION: Cytometer Setup
7. Create a histogram plot with (405 nm) V-SSC-H on the X-Axis and make sure it is plotted on a log-scale.
SECTION: Cytometer Setup
8. Set the cytometer triggering threshold to (405 nm) V-SSC-H. All samples should be acquired with the lowest flow rate, typically ~10-15 µL min-1.
 

SECTION: Cytometer Setup
9. Acquire the DPBS tube while viewing the histogram plot from  . Adjust the detector gain or trigger threshold until the instrument noise is being acquired at ~1000 events/sec. The instrument noise floor is distinct from detected background events in sheath as it has a sharp increase. In a system with debris there may be a tail that elongates out of this this sharp peak. 
 
  

SECTION: Cytometer Setup
9.1. Recording this noise is not necessary as this step is identifying optimal settings.
SECTION: Cytometer Setup
10. Acquire the 'Beads' tube from   . Using the plot from    adjust the FSC and B-SSC gain until the single bead population is clearly visible and can be easily gated from the doublet population to the top right of it. Use the Violet SSC trigger settings identified in   
 
 
  

SECTION: Cytometer Setup
11. Creating a gate around the single bead population named 'Bead Gate';
SECTION: Cytometer Setup
12. Adjust the stopping criteria of the instrument to record until 10,000 events are acquired on 'Bead Gate' drawn in   .

SECTION: Performing Voltration
13. Voltration can now be performed by recording the 'Beads' tube at multiple fluorescent detector gains, leaving the trigger threshold and light scatter gains consistent. It is recommended that a recording of at least 10 fluorescent detector settings is taken. Including more increments within a voltration will result in being more confident of the subsequent optimal detector settings.  
 

  

SECTION: Performing Voltration
13.1. To ensure accurate data analysis, the brightest bead must be visible on at least two of the selected gains.
SECTION: Performing Voltration
14. Fluorescent channels can be cross calibrated to determine lower limit of detection for the channel in calibrated units in the FCMPASS software. A cross calibration between the desired MESF bead and QbSure beads should be acquired at a fluorescent detector gain where all MESF bead populations are on-scale and fully resolved from the noise.

 

SECTION: Cytometer Setup
5. On the Cytek Aurora, set window extension to 0. On the CytoFLEX platform turn on 'High Acquisition Mode'.