TITLE: Resource 4: rEV Serial Dilution
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: rEV Reconstitution
1. Briefly centrifuge 100 x g, 5 min, 4 °C rEVs before opening.

SECTION: rEV Reconstitution
2. Add 100 µL of 4 °C deionized water.

SECTION: rEV Reconstitution
3. Pipet up and down to mix. 
 

SECTION: rEV Reconstitution
4. Aliquot 20 µL into low-binding Eppendorf tubes.
 


SECTION: rEV Reconstitution
5. Store at 2-4 °C for up to one day (up to 2 weeks at -80 °C).

SECTION: rEV Dilutions
6. Label 10 low-binding Eppendorf tubes: 

 

SECTION: rEV Dilutions
7. Add 980 µL of DPBS (preferably 0.1 µm  filtered) to the tube 1 from  .
SECTION: rEV Dilutions
8. Add 500 µL of DPBS (preferably 0.1 µm filtered) to the remaining tubes.

SECTION: rEV Dilutions
9. Add 20 µL of the stock reconstituted rEVs to the tube labeled 1 (  ), pipet up and down to mix.

 

SECTION: rEV Dilutions
10. Pipette 500 µL of the 1:50 dilution in tube 1 into the 1:100 dilution in tube 2, pipet up and down to mix.
 


SECTION: rEV Dilutions
11. Repeat the above step for each of the remaining dilution tubes until the serial dilution has been completed for all dilutions.
SECTION: rEV Dilutions
12. Keep on ice during acquisition.

SECTION: Flow Cytometer Acquisition
13. Use the instrument settings that were established from the previous gain incrementation and scatter calibration resources. On the Aurora, set the window extension to 0. On the CytoFLEX, use the high acquisition mode. Collect for at least 1 minute at a low flow rate with a 30 second recording delay if using plate mode.

 

SECTION: Flow Cytometer Acquisition
14. Collect a DPBS sample in a FACS tube for one minute on low.
 


  

SECTION: Flow Cytometer Acquisition
15. Collect all rEVs samples for one minute on low, starting with the highest dilution (least concentrated) and working down to lowest dilution (most concentrated). This will reduce the effect of carry over from high concentrations. 
  

SECTION: Flow Cytometer Acquisition
17. 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. Run QbSure beads on low and collect 10,000 bead events.

 

 
  
SECTION: Flow Cytometer Acquisition
16. Run a DPBS sample in a FACS tube to make sure there are no carryover events; there should be no events in the B1 and B2 channel. 
 

SECTION: Flow Cytometer Acquisition
19. 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: Flow Cytometer Acquisition
18. Run the 100 nm polystyrene NIST beads at the same settings as the rEVs (diluted in DPBS to a concentration of 5E6 p/mL) until at least 10000 bead events have been acquired.

 

SECTION: Flow Cytometer Acquisition
13.1. Set your instrument to trigger on the most sensitive side scatter channel at the FCMPASS optimal gain output as determined by the FluoSpheres scatter voltration. Set the threshold in the noise such that the event rate is ~1000 events/sec when running DPBS.
 

SECTION: Flow Cytometer Acquisition
13.2. Set the FITC parameter to the FCMPASS optimal gain output as determined by the QbSure fluorescent voltration.