Culture DiFi cells11 under the appropriate culture conditions as previously

published. BeWo cells were cultured as published earlier12,13 with the

modification of addition of 10% exosome-depleted FBS).

Culture placental explant tissue14 and adipose tissue under the appropriate

culture conditions as previously published (see Notes 1 and 2).

If performing tissue culture, collect conditioned culture media supernatant

along with all small pieces of tissues into a fresh 15 mL or 50 mL Falcon

tube.

2000x g,0h 0m 0s Centrifuge at 2,000 xg for 10 minutes at Room temperature (20-22°C).

Remove conditioned culture media supernatant carefully without disturbing

the pellet and pass it through 0.8 μm syringe filter (optional) to avoid any

cellular debris contamination (See Note 3).

Aliquot and store filtered media stored at -80°C.-80°C

Size Exclusion Chromatography to Isolate Extracellular Vesicles

Turn on the Izon Automatic Fraction Collector (AFC) by switching on the

power button at the rear of the instrument

Set up the 35 nm qEV Original Gen 2 column on the column mount. Insert

the column from above into the column mount, then remove the lower cap and carefully dock the column. Ensure that the IZON logo on the column is

facing away from the AFC tower.

Once the qEV column and the column mount have been attached to the

AFC, the column type is determined automatically and is displayed on the

AFC touchscreen.

From the screen, set the fraction number to 4, fraction size 400 µL, and

buffer volume to 2.9 mL for collection ( See Note 4)

Lock the collection tubes onto the carousel in the appropriate orientation.

Gently place the carousel onto the carousel plate (See Note 5).

Add 500 µL of the sample on the top of the qEV original column Gen 2

Collect fractions 7 to 10 (position 1 to 4) and combine them. The volume

can be adjusted by concentrating them according to the downstream

experiments. (See Note 6).

Differential Ultracentrifugation to Isolate Extracellular Vesicles and Non-Vesicular Extracellular Particles s

Centrifuge tissue or cell culture conditioned media in 15 mL tubes at 2,000

xg for 20 minutes at4°C to remove dead cells. Discard the pellet and

transfer the supernatant to a new collection tube

Optionally, pellets can be washed by resuspending them in PBS and

repeating the corresponding centrifugation step.

To isolate large extracellular vesicles (l-EVs), centrifuge the supernatant at

10,000 xg for 40 minutes at4°C

Resuspend the pellet containing l-EVs in PBS-H and store at -80°C until

further analysis (See Note 7). The supernatant is transferred to the

corresponding ultracentrifuge tube.

To isolate small extracellular vesicles (s-EVs) centrifuge the supernatant at

167,000 xg for 4 hours at4°C

Resuspend the pellet containing s-EVs in PBS-H and store at -80°C until

further analysis. The supernatant is transferred to the corresponding ultracentrifuge tube

To isolate exomeres, centrifuge the supernatant at 167,000 xg for 16 hours

at4°C

Resuspend the pellet containing exomeres in PBS-H and store at -80°C

until further analysis. The supernatant is transferred to the corresponding

ultracentrifuge tube.

To collect supermeres, centrifuge the supernatant at 367,000 xg for 16

hours at4°C .

The pellet containing supermeres is resuspended in PBS-H and stored at -80°C until further analysis.

Vesicle Flow Cytometry to Characterize Extracellular Vesicles in Conditioned Culture Media - Determining optimum dilution for the samples for vFC assay

Thaw neat, unconcentrated conditioned culture media samples on ice (See

Note 8).

The .fcs files generated by the flow cytometer can be analyzed in the FCS

Express software package (See Note 15).

The dilution that generates 25,000-50,000 total events is considered the

optimum dilution for the vFC assay.

Vesicle Flow Cytometry to Characterize Extracellular Vesicles in Conditioned Culture Media - Staining of Optimally Diluted EVs with Fluorescently Labelled Antibodies Staining of Optimally Diluted EVs with Fluorescently Labelled Antibodies

Thaw neat, unconcentrated conditioned culture media samples on ice

The total staining reaction volume should be 50 µL .

• For single surface marker staining, add 5 µL of pre-titrated antibodies

to each tube of the reaction mixture.

• Add 5 µL of 10X vFRed to each tube of the reaction mixture.

• The sample input must be calculated as per the optimal dilution decided

in the previous experiment.

The total vFC staining/dilution buffer needs to be adjusted as per the

volume of sample input, total volume of antibody/antibodies, and the

volume of vFRed added to the reaction mixture

After the total reaction mixture is made, pipette the contents of the well to

mix using a 50 µL multichannel pipette

Incubate the staining reaction mixture in dark for 1 hour at room

temperature.

Post-stain, dilute the sample 1000X in vFC dilution/staining buffer, (See

Note 12 and Note 13), pipette 4-5 times to mix the wells after the dilutions

are made using a 1000 µL multichannel pipette. The sample dilutions are

ready to be read using the Cytoflex S.

Before loading the sample into the flow cytometer, clean the cytometer by

running a cleaning reagent followed by a 0.22 µm-filtered deionized water

wash.

Prepare vFRed 10X working solution from the 100X stock using vFC buffer.

(See Note 9)

Open a new experiment in the flow cytometer using a pre-saved template

used for the assay (See Note 14)

Analyze the .fcs files generated by the flow cytometer using the FCS

express software package (See Note 15) to calculate the diameter of the

vesicles and the number of vesicles that are positive for each surface

marker, and each combination of surface markers (Figure 1a-c).

Citation

Figure 1: Representative images showing CD9+ extracellular vesicles inneat, unconcentrated conditioned culture media using vesicle flow cytometry(vFC). (A) ⁺ extracellular vesicles inneat, unconcentrated conditioned culture media using vesicle flow cytometry(vFC). (A) DiFi conditioned culture mediastained with CD9 showing 58.6% CD9⁺ EV population, (B) Placentalexplant supernatant (PES) showing 1.6% CD9⁺ EV population, and (C)BeWo conditioned culture media showing 41% CD9⁺ EV population.

Make serial dilutions of the samples: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320.

(See Note 10). Make these dilutions in individual wells of a 96-well plate.

Add 5µL of 10X vFRed to each of the sample dilution wells and pipette mix

them using a 200µL. (See Note 11).

Incubate the 96-well plate in the dark for one hour at room temperature

Post incubation, dilute the sample 1:1000 in vFC dilution/staining buffer.

(See Note 12 and Note 13) and mix the wells 4-5 times after the dilutions

are made using a 1000 µL multichannel pipette. The sample dilutions are

ready to be read using the Cytoflex S (or other suitably sensitive flow

cytometer).

Before loading the sample into the cytometer, clean the flow cytometer by

running 250µL the Coulter-cleanse cleaning solution followed by 250µL of

a 0.22µm-filtered deionized water wash.

The sample can be loaded into the flow cytometer in two ways: a) manually:

by using FACS tubes/Ria vials for individual samples; or b) semiautomated using a 96-well plate-loader. The semi-automatic plate loader

can be used to load the samples into the flow cytometer since the number

of sample tubes is >10.

Open a new experiment in the CytExpert software using a pre-saved

template used for the assay (See Note 14).

Microfluidic Resistive Pulse Sensing to Characterize Fractions Collected from Size-Exclusion Chromatography y

Begin by priming the microfluidic system of the Spectradyne nCS1 with

PBS-T 0.1%.

Filter the diluent using the 0.02µm syringe filters to remove any particulate

matter that could cause false-positive counts.

Dilute the EV samples 1:100 in the prepared PBS-T 0.1% buffer to align

with the sensitivity range of the TS-400 cartridge.

Load 7 µL of the diluted sample into the cartridge. Ensure the sample is

applied smoothly to prevent air bubbles, which can affect the measurement

accuracy

Start the measurement process. The nCS1 system will automatically adjust

the pressure and voltage settings to optimize the detection of particles

within the sample

Continue data acquisition until the standard error of the mean particle count

is less than 2%, indicating reliable data collection. This may require

continuous acquisitions over several minutes.

Upon completion of the measurements, the data collected are processed

using the nCS1 Data Viewer software.

Apply peak filters and perform background subtraction as directed by the

manufacturer to ensure accurate particle size distribution is obtained

(Figure 2).

Citation

Figure 2: Representative image showing size range of placental extracellularvesicles isolated using size exclusion chromatography (SEC) from placentalexplant supernatant (PES). Microfluidic resistive pulse sensing conducted using theSpectradyne nCS1 shows that the size range of EVs isolated from Amiconconcentrated PES ranges from 75-100nm in size.

Immunoblotting to Characterize Extracellular Vesicles and NonVesicular Extracellular Particles

Lyse EVs and NVEPs purified using SEC or ultracentrifugation using 20 uL

of 10X RIPA buffer (fractions are collected on 200 uL of PBS-H) containing

protease and phosphatase inhibitors.

Wash membranes 3 times,10 minutes each in TBS-T shaking at RT.

Prepare the substrate working solution by mixing equal parts of the

Substrate and Stable Peroxide components. Use a sufficient volume to

ensure that the blot is covered with the substrate and the blot does not

become dry

Incubate the membrane with the substrate working solution for 5 minutes.

Image the blot on an iBright Imaging system (Figures 3 and 4).

Citation

Figure 3: Representative images of adipose tissue-derived extracellularvesicles isolated using size exclusion chromatography (SEC) characterized byimmunoblotting. Representative Western blot of the expression ofAlix, TSG101, CD63, CD9, and CD81 as determined in pooled adiposetissue-derived EVs isolated by SEC.

Citation

Figure 4: Representative images of adipose tissue-derived extracellularvesicles and non-vesicular extracellular particles (NVEPs) characterized byimmunoblotting. Representative Western blot of the expression ofAGO2, CD63, and CD9 as determined in adipose tissue-derived EV and NVEP samplesisolated using ultracentrifugation.

To remove debris, centrifuge lysates at 14,000 xg for 15 minutes and

collect and store supernatants at -80°C (up to 6 months).

Prepare BSA standards, and store up to 2 weeks at 4°C.Conduct BCA

assay according to manufacturer’s instructions.

Prepare 30 μg of sample lysate diluted in 4x Laemmli sample buffer and

heat them at 95 °C for 5 min (closed cap, to avoid evaporation which might

reduce the load volume). Load protein ladder (5 μL) and 30 μg of samples

on 4–20% Criter^TMon‱ TGX Stain-F^TMee‱ precast gels

Perform electrophoresis at a constant 120 V for 1 h in 1x Tris/Glycine/SDS

running buffer

Following electrophoresis, transfer proteins onto PVDF membranes using

a Bio-Rad Trans-Blot Turbo Transfer System

. Block the membrane in TBS-T+5% BSA for 1h shaking at RT.

Incubate membranes with primary antibodies (15 mL- 1:1000, or other

optimized dilution) in TBS-T+5% BSA overnight shaking at 4°C.

Wash membranes 3 times in TBS-T (20 mL for 10 minutes shaking at RT.

Incubate membranes with secondary antibodies at optimized dilution

in TBS-T+5% BSA for 1 hour, shaking at RT

1. We recommend using tissue as soon as possible after harvest to avoid cell death

which might interfere with the EV and NVEPs preparation

2. It is critical to pay attention to the amount of medium/weight of sample to maintain

a constant ratio for consistency and reproducibility.

3. It is critical to collect the culture supernatant without disturbing the pellet as the

pellet might have contaminating red blood cells.

4. It is important to follow the manufacturer’s default settings for the Izon AFC.

5. Ensure that the carousel alignment hole engages with the raised carousel alignment

pin on the carousel plate. Once the carousel has been loaded, the instrument will

pass through the column preparation steps before initiating the collection.

6. Before starting the collection, all collection parameters should be reviewed properly

which include default buffer volume, position of fractions and volume of fractions to

be collected

7. It is recommended to store the pellets in PBS-H to improve sample preservation 15

8. Repeated-freeze thaw cycles can negatively affect samples. Therefore, the sample

should be aliquoted to avoid multiple freeze-thaw cycles.

9. For vFC, the vFRed dilution/staining buffer and stock solution should be brought up

to room temperature before making the working solution of the 10X vFRed. Detailed

Notes for vFC, including Protocols for instrument calibration, sample preparation

and staining, flow cytometry, and data analysis are available on the Cellarcus web

site (www.cellarcus.com).

10. Make a plate map for preparing the sample dilutions.

11. Set the 200 μL multichannel pipette to 45 μL and mix the solution slowly for 4-

5 times. Do not fix the pipette to 50 μL (total reaction volume) because it may

introduce bubbles.

12. After staining, a post-stain dilution is performed to reduce background from

unbound stain. For plate-based assays where well capacities are ~300 uL, the

1000X post-stain dilution is performed in two steps. After incubating the staining

mixture in dark conditions for 1 hour at room temperature, 8µL of staining mixture

is mixed with 237 µL of vFC staining/dilution buffer (Dilution-1). After pipette mixing

the contents of Dilution-1 well, 8µL of Dilution-1 is mixed with 237 µL of vFC

staining/dilution buffer (Dilution-2). Dilution-2 is read by the cytometer

13. This protocol has been adapted for use with the CytoFlex instrument in the

authors’ lab. For other instruments, detailed protocols are available on the Cellarcus

web site (www.cellarcus.com).

14. The CytoFlex cytometer acquisition settings are as follows:

Scatter gains (VSSC, SSC, and FSC) = 500

Active channels = All channels

Primary threshold =vFRed-H

Manual Threshold = 1900

Width parameter = vFRed (690/50)

Time to record = Checked, 120 seconds

Events to record = Unchecked

Events to display = 50,000

Sample flow rate = Fast (60 µL/min)

15. The instrument setup Protocols (www.cellarcus.com) use calibration particles

to perform spectral and intensity calibrations, which are stored as “.comp” (spillover

and compensation matrices for the fluorophores being measured), “.cal”

(expressing fluorescence in units of molecules per particle), and “fcf” files (used for

calibration of EV size [surface area, diameter, etc], scattering cross-section, and

fluorophore surface density) which are loaded in the data analysis Layout to apply

these calibrations

16. For Spectradyne analysis, in this protocol, we used a TS-400 cartridge. These

cartridges are designed for analyzing particles ranging from 65 to 400 nm, suitable

for small extracellular vesicles. If the EVs are collected via a different method and

thus may be larger in diameter, different cartridges are recommended (e.g., C900/TS-900 for 130-900 nm particle diameters, or C-2000/TS-2000 for 250-2000

nm particle diameters.