08 Labeling Chemistry

ExoSparkler Exosome Protein Labeling Kit-Red

Labeling Chemistry

Exosome Protein Fluorescent Staining

Observe exosome dynamics accurately without extracellular aggregation
Cover steps from fluorescence labeling to purification

Product code

EX05　 ExoSparkler Exosome Protein Labeling Kit-Red

Unit size	Price	Item Code
5 samples		EX05-10

*Protein amount : 1-10μg/ sample, Particle count : 10 to 100 x 10⁸ /samples
(As purified exosome using ultracentrifugation)

Component

5 samples	・Protein Dye-Red ・Filtration tube	×1 ×5

C of A

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Description

Exosomes are a form of extracellular vesicle (EV), which may 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 suitable labeling target. ExoSparkler series can be used for staining of purified exosomal membrane or protein, which allows imaging of labeled exosomes taken up by cells.

Manual

Manual

Technical info

ExoSparkler series contains filtration tubes available for the removal of dyes unreacted after fluorescence labeling, as well as an optimized protocol for labeling exosomes. Our ExoSparkler series makes it possible to prepare fluorescence labeling of exosomes using the simple procedure.

ExoSparkler series product comparison

Experimental conditions
Exosomes were purified by ultracentrifugation (10μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (1.25×10⁴ cells), and the cells were incubated for 24 hours. Cells were washed, and immunofluorescence images showing labeled exosomes were observed.

Detection conditions
Green: Ex 488nm/Em 490-540nm
Red: Ex 561nm/Em 570-640nm
Deep Red: Ex 640nm/Em 640-760nm

Localization of exosomes

Exosomes stained with ExoSparkler Exosome Protein Labeling Kit-Deep Red（Item#: EX06, Protein Dye-Deep Red）or ExoSparkler Exosome Membrane Labeling Kit-Red（item#：EX02, Mem Dye-Red）were added to HeLa cells, and the cells were co-stained with lysosome staining reagent (Green).

Experimental conditions
Exosomes were purified by ultracentrifugation (5μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (0.75×10⁴ cells), and the cells were incubated for 3.5 hours. After that, cells were stained with lysosome staing reagent (Green). Cells were washed, and immunofluorescence images showing labeled exosomes were observed.

Detection conditions
Protein Dye-Deep Red (purple) : Ex 640nm/Em 640-760nm
MemDye-Red (Red) : Ex 561nm/Em 570-640nm
Lysosome staing reagent (Green). : Ex 488nm/Em 490-540nm

Q & A

Q Are there recommended purification methods for exosome?: A

We recommended the exosomes prepared by ultracentrifuge. We also have experience labeling the exosomes purified by immunoprecipitation and magnetic beads.
　You can also use the ExoIsolator Exosome Isolation Kit (EX10), which has been proven the recovery rate equivalent to that of the ultracentrifugal method.
　
In addition, exosomes prepared by the polymer-based precipitation method are not applicable for the following reasons.
1) The polymer may clog the filtration tube.
2) The polymer may interact with exosomes and change the membrane potential of exosomes. If the membrane potential changes, the intracellular kinetics of labeled exosomes may change when they are used in cellular uptake assays.

Q Some dyes remains on the filte after purification. Have unlabeled dyed been removed?: A

Unlabeled dye is likely to be retained inside the filter, but we have confirmed that the unlabeled dye is not remaining on top of the filter. Please refer to the following experiment:

① We stained the 10 µg exosomes (as protein amount) purified by ultracentrifugation.
② We also prepared only using buffer as a control
3. The recovered solution is added to HeLa cells (1.25 x 10⁴ cells), and the fluorescence image was observed 4 hours later.
As a result, it was confirmed that no bright fluorescent particles (unlabled dyes) were observed in the cells using the recovered solution with the only buffer.

① Exosome + Buffer

② Only Buffer

Fluorescent images at 4 h incubation

Detection conditions
Green：Ex 488 nm / Em 490 – 540 nm
Red ：Ex 561 nm / Em 570 – 640 nm
Deep Red：Ex 640 nm / Em 640 – 760 nm

Q Can I store the exosomes after it has been stained?: A

We can not recommend to store the exosomes after it has been stained.
Please use the stained exosomes as soon as possible.

Q Which media have been used in exosome uptake experiments?: A

We had evaluated the exosome uptake experiments using MEM (Minimum Essential Medium) and DMEM (Dulbecco’s Modified Eagle’s Medium). We can not recommend to use the serum-free medium because this kit has been optimized to observe in serum-containing medium.

Q What is the amount of exosomes that can be labeled per sample?: A

Isolated exosomes (ultracentrifugation method), protein: 1-10 μg/sample, number of particles: 10-100x 10⁸ particles/sample.

Q If there are contaminants other than exosomes, are they labeled by ExoSparkler, too?: A

Yes, if there are contaminants in the sample, such as proteins or polymers*, they will be labeled by ExoSparkler too. Therefore, please prepare purified exosomes.
For details on the purification method, please refer to the FAQ "Are there recommended purification methods for exosomes?"

*Polymers may remain depending on the purification method.

Handling and storage condition

Handling and storage condition
0-5°C、Protect from moisture

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ExoSparkler Exosome Protein Labeling Kit-Red

ExoSparkler Exosome Protein Labeling Kit-Red