TITLE: Resource 6: rEV Fluorescent Detector Setting Incrementation
AUTHORS: Sean M Cook, Vera A. Tang, Joanne Lannigan, Jennifer Jones, Joshua A Welsh

[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 Preprepation
1. Briefly centrifuge rEVs at 100 x g, 5 min, 4 °C before opening
SECTION: Sample Preprepation
2. Add 100 µL of 4 °C deionized water to rEV vial. Pipette up and down to mix well.


 

SECTION: Sample Preprepation
3. Create 5 mL of a 1:2000 dilution of rEVs by pipetting 1 µL  rEVs into 1999 µL  DPBS in a low-binding Eppendorf tube. This gives a 5E6 p/mL solution.
SECTION: Cytometer Setup
6. 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
8. Create a histogram plot with the FITC parameter (CytoFLEX: FL1 | B-525-40, Aurora: B2 | B-524.5-17) on the X-Axis and make sure it is plotted on a log-scale.
SECTION: Cytometer Setup
9. 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
10. Set the cytometer triggering threshold to the violet SSC parameter at the same settings used to acquire the bead fluorescent voltration. All samples should be acquired with the lowest flow rate, typically ~10-15 µL min-1.
SECTION: Cytometer Setup
11. Acquire the 'DPBS' tube while viewing the SSC 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
11.1. Recording this noise is not necessary as this step is for identifying optimal settings.
SECTION: Sample Preprepation
4. Label a FACS tube as 'DPBS'. Pipette 500 µL DPBS into the tube.

SECTION: rEV Acquisition
12. Validation of FCMPASS outputs can now be performed by acquiring the rEVs at the same voltration gains used when acquiring the QbSure fluorescence voltration. 

 

  

 

SECTION: rEV Acquisition
13. Pipette 200 µL from the 5E6 rEV solution from   into a FACS tube. Acquire the FACS tube for 60 s on the lowest flow rate at one of the fluorescence gains. Repeat this for each gain.

 

SECTION: rEV Acquisition
16. If calibration of data into standard units is desired, the 100 nm polystyrene NIST bead and QbSure beads can be used to calibrate data by following the FCMPASS experiment calibration protocols.
 


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

SECTION: Sample Preprepation
5. Label as FACS tube as 'QbSure', and add 500 µL DPBS. Vortex the QbSure beads for 5 sec and add 3 drops QbSure beads into FACS tube.
SECTION: rEV Acquisition
15. Run QbSure beads on low at the same fluorescent settings and collect 10,000 bead events for each gain.

 

 

SECTION: rEV Acquisition
14. Run the 100 nm polystyrene NIST beads at the same scatter settings as the rEVs (diluted in DPBS to a concentration of 5E6 p/mL) until at least 10000 bead events have been acquired. The 100 nm bead only needs to be acquired once at the scatter settings used in the experiment.