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Supplementary Protocols

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Lipid/Protein Analysis | Relative Quantitation of EVs | Surface Phenotyping | Detecting Protein Cargo

Direct Detect® IR Spectrometer Analysis of Lipids and Proteins in EV samples

Many traditional protein assays are not suitable for analyzing extracellular vesicle preparations because the presence of lipids and other nonprotein components in the sample may interfere with protein analysis. For this reason, IR-based, assay-free quantitation afforded by the Direct Detect® system is particularly well-suited to monitoring EV enrichment.

Summary: samples are lysed, and 2 µL of the lysate is used to determine protein and lipid content. Below is a protocol we have used in our laboratories to perform this analysis.

1. In our laboratory, we perform EV lysis using
or
Supplemented with protease inhibitors (for example, Cat. No. 539134 Protease Inhibitor Cocktail set III, EDTA-free).
Lysis is then performed either of two ways:
  • Directly on EV sample
  • On bead-captured EVs (see below for bead capture protocol)
2. Lysates are then quantitated using the Direct Detect® protocol for lipids analysis and the Direct Detect® protocol for protein quantitation

Bead-based EV Capture and Relative Quantitation Using a guava easyCyte™ Flow Cytometer

Performing bead-based relative quantitation of your extracellular vesicle preparation step involves three steps:

I. Fluorescent labeling of EV prep
  1. Combine 50 µL EV sample with 50 µL PBS – 100 µL total reaction volume
    NOTE: 50 µL represents approximately 10% of recovered sample (10 mL cell culture supernatant concentrated to 500 µL)
  2. Add 1 µL 1 mM BODIPY® FL C5-ceramide (Thermo Fisher Cat. No. D3251) – final dye concentration is 10 µM.
    NOTE: BODIPY® FL C5-ceramide is a green fluorescent dye (Ex/Em 503/512 nm). It is detected in the GREEN channel on guava easyCyte™ flow cytometers.
  3. Incubate at 37°C for 30 minutes.
  4. Remove free dye by buffer exchange using Amicon® Ultra 0.5 filter 3K MWCO (Cat. No. UFC500396).
  5. Load sample into assembled device.
  6. Add 400 µL PBS, centrifuge at 14,000 x g for 15 minutes. Aspirate filtrate from collection tube.
  7. Repeat step 6.
  8. Recover sample from the device by reverse spin (1000 x g for 2 min). Recovered volume should be approximately 50 µL.
  9. Store sample at 4 °C protected from light.


II. Label PureProteome™ Streptavidin Magnetic Beads with biotinylated antibody
NOTE: We recommend using round bottom 1.5-2 mL microfuge tubes for the following steps.
  1. Each bead labeling reaction contains 5 µL bead slurry and 5 µg biotinylated antibody [For example, Anti-human CD63-biotin, (Clone H5C6, BioLegend Cat. No. 353017)].  If the same antibody is to be used for multiple labeling reactions, the labeling can be performed in bulk.
  2. Wash 5 µL PureProteome™ Streptavidin magnetic bead slurry (Cat. No. LSKMAGT10) with 500 µL PBS, place on PureProteome™ magnetic stand (Cat. No. LSKMAG08) for 1 minute, aspirate and remove tube.
  3. Perform step 2 two more times.
  4. Resuspend beads in 5 µg biotinylated antibody, bring reaction to 100 µL with PBS.
  5. Incubate with back-and-forth rocking for 30 minutes at room temperature.
  6. Add 400 µL PBS, mix by pipetting, and place tubes on magnetic stand for 1 minute. Aspirate.
  7. Perform step 6 two more times.
  8. Resuspend beads in 100 µL PBS.
  9. Optional – If multiple reactions were prepared in bulk, resuspend beads in a volume of 100 µL per 5 µL beads. Aliquot 100 µL to use in each EV capture reaction (see section III below).


III. Bead capture of CD63+ EVs
  1. Combine antibody-bead complexes (100 µL) with BODIPY®-labeled EV prep and bring volume to 200 µL.
  2. Incubate at 4 °C overnight with gentle back-and-forth rocking. Keep protected from light.
  3. Add 300 µL PBS, mix by pipetting, and place tubes on magnetic stand for 1 minute. Aspirate.
  4. Perform step 3 two more times.
  5. Resuspend beads in 200 µL PBS. Transfer samples to a guava easyCyte-compatible 96 well microplate for acquisition
  6. Run samples on guava easyCyte™ flow cytometer.
  7. Use Forward Scatter vs. Side Scatter Plot to gate on beads as shown:

    Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/forward-scatter-vs-side-scatter-plot.png
    guava easyCyte™ flow cytometer: Acquisition and analysis parameters


  8. Use histogram plot (GREEN-Log) to determine MFI signal (CD63+ EV content) for a given prep.
  9. Compare to control (unlabeled beads) to determine relative EV content per sample.

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Bead-Based EV Capture and Surface Phenotyping Using a guava easyCyte™ Flow Cytometer

  1. Label PureProteome™ Streptavidin Magnetic Beads with appropriate biotinylated antibody as described above.
  2. Capture EV as described above.
    NOTE: There is no need to BODIPY®-label EVs for this analysis
  3. Add fluorescent-labeled antibodies. Mix by pipetting. Incubate at 4'C for 45 1 hour with gentle back-and forth rocking. Keep protected from light.
    NOTE: Please consult the guava easyCyte™ flow cytometer page for a list of compatible fluorescent dyes
  4. Add 300 µL PBS, mix by pipetting, and place tubes on magnetic stand for 1 minute. Aspirate.
  5. Perform step 11 two more times.
  6. Resuspend beads in 200 µL PBS. Transfer samples to a guava easyCyte-compatible 96 well microplate for acquisition

Detection of Protein Cargo Using the SNAP i.d.® Western Blot Probing System

Unlike conventional Western blot probing, where diffusion is the primary means of reagent transport, the SNAP i.d.® 2.0 system uses a vacuum to actively drive reagents through the membrane.

For best results, systematically vary antibody concentrations the first time you try to detect your EV protein cargo using the SNAP i.d.® system, as the conditions may differ slightly from traditional, diffusion-dependent Western blot probing. Also, carefully read and follow the SNAP i.d.® User Guide, which includes antibody concentration optimization guidelines.

The summary of the protocol is included here:

  1. Lyse EV sample as in the Direct Detect® protocol. Separate proteins using SDS-PAGE and transfer to an Immobilon®-P membrane.
  2. Assemble the blot in the SNAP i.d.® blot holder. Connect SNAP i.d.® system to vacuum trap and vacuum pump, with inline filter in place. Insert blot holder.
  3. Apply 30 mL of blocking solution; for example, 0.5% nonfat dry milk in TBS-T.
  4. Turn on vacuum. When frame is empty of blocking solution, turn vacuum off.
  5. Apply appropriate amount of primary antibody to the blot holder. Incubate 10 minutes. (Longer if deemed necessary during optimization).
  6. Turn on vacuum. Turn off when frame is empty of antibody solution.
  7.  Wash 4 times by applying 30 mL of TBST and applying vacuum to remove wash solution.
  8. Apply secondary antibody for 10 minutes.
  9. Apply vacuum to remove.
  10. Wash 4 times by applying 30 mL of TBST and applying vacuum to remove wash solution.
  11. Remove blot holder.
  12. Apply detection reagents (such as Luminata™ chemiluminescence detection reagents).
  13. Detect signal using x-ray film or other method.

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