TITLE: Resource 7: rEV immunophenotyping
AUTHORS: Vera A. Tang, Sean M Cook, 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 Preparation
1. Briefly centrifuge the rEVs 100 x g, 5 min, 4 °C before opening
SECTION: Sample Preparation
9. In the same 96-well V-bottom plate from  , add 10 µL 1:5 rEV solution to wells B1-G3. Add 10 µL DPBS to wells A1-G1. Add 10 µL from the PE working solution tubes to wells B1-G1, add 10 µL from the APC working solution tubes to wells B2-G2, add 10 µL from the PB working solution tubes to wells B3-G3.

SECTION: Sample Preparation
10. In the same 96-well V-bottom plate from  , add 10 µL from the corresponding PE working solutions tube to wells B4-G4. Add 10 µL from the corresponding APC working solutions tube to wells B5-G5. Add 10 µL from the corresponding PB working solutions tube to wells B6-G6. These wells will serve as the DBPS+AB control wells.

SECTION: Cytometer Setup
16. Setup cytometer to acquire at FCMPASS output gains for both light scatter and fluorescent detectors as determined by the FCMPASS detector optimization module outputs. For the Aurora, an optimal gain template is returned to import into the Spectraflo software.
SECTION: Cytometer Setup
19. Acquire all wells for at least 60 sec.
SECTION: Sample Preparation
12. Using a separate 96-well V-bottom plate, add 199 µL DPBS into wells A1-G6. Pipet 1 µL from the incubation plate into the new plate in the same wells and reverse pipette to mix.

 

SECTION: Cytometer Setup
22. 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: Sample Preparation
6. In a 96-well V-bottom plate, add 10 µL 1:5 rEV solution to wells A1-A3 
SECTION: Sample Preparation
11. Cover and incubate this plate for 30 min at RT.
SECTION: Sample Preparation
14. Re-cover incubation plate and let incubate ON at RT to repeat measurements next day as directed in the following steps.
SECTION: Sample Preparation
7. Add 10 µL DPBS to wells A1-A3 and reverse pipet to mix. These wells will serve as the rEV controls

SECTION: Sample Preparation
8. Add 20 µL DPBS to wells A4-A6. These wells will serve as buffer only controls
SECTION: Sample Preparation
13. Add 200 µL DPBS into wells A7-G7. These wells will help reduce sample carryover after each rEV+AB combination is acquired.
SECTION: Cytometer Setup
17. On the Cytek Aurora, set window extension to 0. On the CytoFLEX platform turn on 'High Acquisition Mode'.
 

SECTION: Cytometer Setup
18. Set the cytometer triggering threshold to the violet SSC parameter and run a DPBS control first to ensure the cytometer is clean and thresholds/event rates have remained unchanged. The background event rate should be ~1000 events/sec. All samples should be acquired with the lowest flow rate, typically ~10-15 µL min-1.
SECTION: Cytometer Setup
20. In an open well, 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: Cytometer Setup
21. In an open well, add 200 µL from the QbSure FACS tube and collect 10,000 bead events at the same settings as the rEVs.
 

 

SECTION: Sample Preparation
15. 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: Cytometer Setup
23. Don't forget to repeat the acquisition for the samples incubating ON.
SECTION: Sample Preparation
4. Prepare the antibody dilutions for a 2x staining concentration of each of the antibodies to be used in the antibody titration. See the example below for sample calculations prepared for a staining concentration of 2 µg/ml. 
 
            Ab Stock Concentration (ug/ml)      2x staining conc (ug/ml)      Stock volume for 2x staining conc (uL)      Total volume for 2x staining conc   (uL)      Volume of PBS to be added (uL)        anti-Human CD81-PE (5A6)      120      4      2      60      58        anti-Human CD81-APC (5A6)      200      4      2      100      98        anti-Human CD81-PB (5A6)      300      4      2      150      148   
 
SECTION: Sample Preparation
2. Add 100 µL of 4 °C deionized water. Pipette up and down to mix.
 


SECTION: Sample Preparation
3. Dilute the reconstituted rEVs 1 in 5 in PBS for staining* Take 50 µL of the rEV stock and add 200 µL of PBS.

 

SECTION: Sample Preparation
5. Samples will now be prepared using the following plate map. 
 
            CD81-PE      CD81-APC      CD81-Pacific Blue      CD81-PE      CD81-APC      CD81-Pacific Blue                      rEV      rEV      rEV      PBS      PBS      PBS               0ug/ml      A1      A2      A3      A4      A5      A6               0.0625ug/ml      B1      B2      B3      B4      B5      B6               0.125ug/ml      C1      C2      C3      C4      C5      C6               0.25ug/ml      D1      D2      D3      D4      D5      D6               0.5ug/ml      E1      E2      E3      E4      E5      E6               1ug/ml      F1      F2      F3      F4      F5      F6               2ug/ml      G1      G2      G3      G4      G5      G6      QbSure               PBS      PBS      PBS      PBS      PBS      PBS      100nm PS