ExoSparkler Exosome Membrane Labeling Kit-Green / Red / Deep RedEX01_EX02_EX03 Product Manual

　Recent findings suggest that exosomes, a form of extracellular vesicle (EV), contribute to malignant transformation and the metastasis of cancer. Consequently, intercellular communication via exosomes is attracting considerable interest in the scientific community. To shed light on such communication, labeling techniques based on fluorescent dyes have been used. Fluorescent dyes that label the cellular membrane are commonly used for exosome labeling because the lipid bilayer in exosomes is a good target for labeling. Dojindo’s ExoSparkler Exosome Membrane Dyes overcome issues with existing membrane labeling dyes, such as (1) self-particle formation¹⁾ and (2) a shift in the size of exosomes to larger particles²⁾. Moreover, three different color options of Dojindo’s dyes (Green, Red, and Deep Red) enable application to experiments using multiple labels, such as investigation of exosome localization in intracellular organelles.

 

	Labeling Dyes	Filtration Tube
EX01 : ExoSparkler Exosome Membrane Labeling Kit-Green	Mem Dye-Green x 1	x 5
EX02 : ExoSparkler Exosome Membrane Labeling Kit-Red	Mem Dye-Red x 1	x 5
EX03 : ExoSparkler Exosome Membrane Labeling Kit-Deep Red	Mem Dye-Deep Red x 1	x 5

Store in a cool (0–5°C), dark, and dry place.

• Dimethyl sulfoxide (DMSO)
• PBS
• Micropipette
• Microtube
• Centrifuge

Preparation of Mem Dye stock solution

Add 10 μl of DMSO to a tube of Mem Dye-Green, -Red, or -Deep Red and dissolve using a vortex mixer.

• Store the Mem Dye stock solution at −20°C.
• To avoid the effect of repeated freeze/thaw cycles, it is recommended that Mem Dye stock solution be aliquoted in small volumes
  (e.g., 2 μl each) and then stored frozen.

Preparation of cells

1. Seed the cells on a dish for assaying. Culture the cells at 37°C overnight in a 5% CO₂ incubator.

Labeling of exosome

Table 1. Recommended amount of exosome

Protein amount	1 – 10 μg
Particle count	10 – 100 x 108

• The above mentioned conditions have been optimized for exosome purification by ultracentrifugation.
  In case any other exosome purification method is used, the amount of exosome should be further optimized.
• Exosome prepared by polymer-based precipitation cannot be applied with these kits.

1. Resuspend exosome in 100 μl of PBS (recommended amounts are described in Table 1.
2. Add 2 μl of Mem Dye stock solution to the exosome solution and mix using a vortex mixer.
3. Incubate at 37°C for 30 min.
4. Transfer the labeled exosome solution to a filtration tube.
5. Centrifuge at 3000 × g for 5 min at room temperature.
6. Add 100 μl of PBS to the filtration tube.
7. Centrifuge at 3000 × g for 5 min at room temperature.
8. Repeat steps 6 and 7.
9. Centrifuge at 3000 × g for 5 min at room temperature.
   • Perform an additional 5 min of centrifugation if solution remains on the membrane.
10.  Add 50 μl of PBS to the filtration tube and carefully pipette about 10 times to recover the labeled exosome.
    Transfer the solution to a new microtube.
    • Do not touch the dyes on the membrane to avoid contamination of excess dyes.

    The dyes remain on the membrane ⇒ Carefully pipette to collect the labeled exosomes without touching excess dyes

Addition of labeled exosomes to the cells

1. Remove the culture medium and wash the cells with PBS once.
2. Add culture medium with serum.
3. Add 1–50 μl of labeled exosome in PBS and incubate at 37°C for 1–24 h in a 5% CO₂ incubator.
4. Remove the supernatant and wash the cells with PBS twice.
5. Add culture medium with serum and observe the sample under a fluorescence microscope.

The effects of exosome amounts and incubation times on the fluorescent signal in HeLa cells.

1. HeLa cells were seeded (1.25 × 10⁴ cells/well) on a μ-slide eight-well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
2. The supernatant was removed and the cells were washed once with PBS.
3. A total of 200 μl of DMEM containing 10% FBS was added to each well.
4. The exosome* (5 or 10 μg of protein, 50 or 100 × 10⁸ particle count) purified by ultracentrifugation (from culture medium of HEK293 adapted to suspension mode) was labeled using ExoSparkler Exosome Membrane Labeling Kits, and was added to each well.
   • The protein and particle concentration were quantified by BSA method and nanoparticle tracking analysis, respectively.
5. The cells were incubated at 37°C for 2 or 4 h in a 5% CO₂ incubator.
6. The supernatant was removed and the cells were washed twice with PBS.
7. A total of 200 μl of DMEM containing 10% FBS was added to each well and the cells were observed using a fluorescence microscope.

1. Dominkus, P. P. et al., Biomembranes, 2018, 1860, 1350 – 1361.
2. Dehghani, M. et al., bioRxiv, 2019, doi:https://doi.org/10.1101/532028