PNExo™ Exosome-Green Tea

Question 1

Do quiescent cells secrete exosomes?

Accepted Answer

Yes, quiescent cells are known to secrete exosomes. Exosomes are small vesicles that are released by various types of cells, including both quiescent (non-dividing) and actively dividing cells. These vesicles are involved in cell-to-cell communication, as they carry a cargo of proteins, lipids, and nucleic acids that can be transferred to recipient cells.

Question 2

What strategies can be employed to standardize mRNA expression within exosomes?

Accepted Answer

You can utilize miRNA genes like miRNA16, miRNA26a, miRNA221, miRNA22, miRNA181a, miRNA181c, miRNA103, miRNA191, let7d, as well as small RNAs (5SrRNA and U6snRNA) that are normally expressed in exosomes.

Question 3

Do you offer items designed for conducting studies on tracking extracellular vesicles?

Accepted Answer

Creative Biostructure offers highly purified fluorescent exosome standards for tracking EVs in fluorescence microscopy experiments. These fluorescent exosomes are labeled with a green dye, ensuring stable fluorescent labeling. They are suitable for various applications, providing a long-lasting and well-visible signal.

Question 4

Why is it challenging to detect exosomes through immunofluorescence?

Accepted Answer

Exosomes are so small that it is difficult to get sufficient antibody binding for their detection via immunofluorescence. However, transmission electron microscopy has been performed on exosomes.

Question 5

What is the ideal exosome yield per cell?

Accepted Answer

Certainly, exosome secretion is indeed influenced by cell death. Therefore, it is highly advisable to utilize healthy and viable cells when studying exosomes. Furthermore, it is worth noting that apoptotic cells tend to release a significant amount of apoptotic vesicles.

Question 6

How can I visualize apoptotic bodies, microvesicles, and the exosome pellet obtained through centrifugation?

Accepted Answer

After centrifugation, it is not possible to distinguish between exosome pellet and apoptotic body pellet. To characterize individual extracellular vesicles (EVs), techniques such as Western blotting (WB), flow cytometry (FACS), electron microscopy, and atomic force microscopy can be used. For larger vesicles including apoptotic bodies, immunofluorescence imaging can be employed. To assess size distribution, nanoparticle tracking analysis and dynamic light scattering can be utilized.

Question 7

Which storage condition (4°C, -20°C, or -80°C) is optimal for preserving conditioned media prior to exosome isolation?

Accepted Answer

While we advise processing fresh samples whenever possible, cell media can be stored at 4°C for up to one week without significant changes observed in exosomes or extracellular vesicles (EVs). For long-term storage, it is recommended to store at -80°C.

Question 8

What is the anticipated concentration of RNA in serum exosomes?

Accepted Answer

Exosomes typically contain minimal amounts of nucleic acids. The concentration of RNA in serum samples is so low that it is not detectable on agarose gel or even by nanodrop spectrophotometry. Creative Biostructure provides sensitive, reproducible methods for exosome analysis, our exosome analysis services make it easy to profile exosome lipids, proteins, and RNAs.

Question 9

Can I use heparin or an EDTA tube to collect blood samples to isolate exosomes from plasma?

Accepted Answer

No. Heparin will significantly impair the downstream RNA assays. EDTA may interfere with the downstream PCR assay. Immediately centrifuge the sample to collect the plasma for exosome isolation.

Question 10

Can I block the release of exosomes from cancer cells?

Accepted Answer

Dimethyl amiloride (DMA), GW4869, and a Glucosyl ceramide synthase inhibitor (di threo-1-1phenyl-2-decanoylamino-3-morpholino-1-propanol) have demonstrated their ability to hinder exosome secretion in various types of cells. Alternatively, suppressing the expression of Rab27 or Rab35 through shRNA knockdown represents an excellent approach for inhibiting exosome release.

Question 11

What causes the elevated levels of exosomal miRNA in plasma compared to miRNA levels in serum exosomes?

Accepted Answer

During the preparation of serum samples, when removing the clot from whole blood, it is likely that exosomes and their associated miRNA are being lost. This could explain the disparity in exosomal miRNA levels between serum and plasma, as plasma samples have a lower chance of vesicle and exosomal miRNA loss.

Question 12

Does cell death affect exosomes?

Accepted Answer

The yield of exosomes can vary depending on several factors, including the cell type, culture conditions, isolation methods, and experimental protocols. Exosome yield is typically reported as the number of exosomes per cell or the concentration in a given volume of culture media. To obtain a more accurate estimate of the exosome yield per cell for a particular experimental system, it is recommended to consult relevant scientific literature or perform pilot experiments using the specific cell type, culture conditions, and isolation methods that you intend to use.

Question 13

How do you prepare exosome samples for western blot analysis?

Accepted Answer

For optimal detection of exosome proteins in western blot analysis, we suggest using non-reducing samples. This means that your samples should not contain reducing agents such as DTT or beta-mercaptoethanol during the boiling step prior to loading onto the PAGE gel. This is because the detection antibodies used for targeting tetraspanins in western blot analysis perform best under non-reducing conditions.

Question 14

Is there a distinct marker that can differentiate between apoptotic bodies and exosomes derived from the same cells?

Accepted Answer

Typical markers of apoptotic bodies such as C3b, Annexin V, and thrombospondin can be utilized to distinguish them from exosomes.

Question 15

What are the negative controls used for exosome isolation?

Accepted Answer

Negative controls are used in exosome isolation experiments to help identify and account for non-exosomal components that may co-purify with exosomes and affect experimental results.The following proteins must not be present in the exosome preparation: Grp94HSP90B1, mitochondria (cytochrome CCYC1), calnexin, Endoplasmic reticulum, Argonaute/RISC complex, Golgi (GM130), and nucleus (histones).

Question 16

Could you explain what Plasma Exosome Standards refer to?

Accepted Answer

Plasma exosome standards are purified lyophilized exosomes isolated from a pool of healthy donors by differential ultracentrifugation and microfiltration.

Question 17

Does the freezing affect the purified exosomes?

Accepted Answer

Fresh exosome samples are ideal but frozen samples at -80°C can be used if they were frozen immediately after isolation, have not been thawed multiple times, and have not been frozen for too long. Exosomal membrane is different from cell membrane and contains phospholipids, sphingolipid, ceramides and tetraspanin proteins (CD63, CD81, CD9) that help maintain its stability. However, freeze-thaw cycles can affect the size and morphology of exosomes, which may impact size determination in studies using techniques such as DLS, NTA, bioassays, and EM.

Question 18

For studying exosomes in serum samples, is the use of DMSO necessary?

Accepted Answer

DMSO is used to protect cell membranes when freezing, but it's not recommended to use it when freezing serum samples at -80°C because the small size of exosomes and liquid content makes it unnecessary.

Question 19

How to determine the protein concentration in exosomes?

Accepted Answer

After lysing the exosomes, you can use the Bradford or BCA methods to detect the protein concentration in them, which will provide the protein concentration amount in μg/μl.

Question 20

Is there any specific exosome biomarker?

Accepted Answer

There is currently no widely agreed upon universal exosome marker. It is strongly advised to use multiple exosomal markers such as CD9, CD81, and CD63 to confirm the exosomes.

Question 21

What is the difference between exosomes and microvesicles?

Accepted Answer

The diameter of exosomes is smaller than that of microvesicles, the diameter of exosomes is between 30-120 nm, while the diameter of microvesicles is between 100-1000 nm. Furthermore, they have different markers and play different roles in genetic exchange and cellular communication.

Question 22

Can I freeze purified exosomes after isolation?

Accepted Answer

Fresh samples are always better than frozen samples. However, samples frozen at -80 °C can also be used, provided they are frozen immediately after isolation, without multiple freeze-thaw cycles and for relatively short periods of time. Freeze-thaw cycles may affect exosome morphology and size determination studies.

Question 23

How to isolate and purify exosomes from tissue samples?

Accepted Answer

Since exosomes are secreted, we recommend performing short-term tissue explant cultures to isolate exosomes from tissues. You can collect cell supernatants and isolate exosomes using a protocol similar to the protocol for exosome isolation from cell culture media.

Question 24

Why specific isolation of exosomes is important?

Accepted Answer

Specific isolation of exosomes is important because exosomes play a crucial role in intercellular communication and can carry biological information, such as proteins and RNA, from the producing cells to the recipient cells. Therefore, the specific isolation of exosomes allows for the study of their contents, which can provide insights into cellular processes and potentially lead to the discovery of new therapeutic targets for various diseases. However, it is also important to note that exosomes can be mixed with other extracellular vesicles, such as apoptotic bodies and microvesicles, making specific isolation necessary to ensure accurate characterization and interpretation of results.

Question 25

What are the methods of exosome quantification?

Accepted Answer

There are several methods for quantifying exosomes, including Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), Enzyme-Linked Immunosorbent Assay (ELISA), Western blotting, Flow cytometry, RNA quantification using real-time PCR, Mass spectrometry (MS). Each method has its strengths and limitations, and the choice of method depends on the specific research question and the available resources.

Question 26

What biomarkers do you use to identify the isolated exosomes?

Accepted Answer

We use a combination of exosomal markers such as Alix 1, CD9, CD63, CD81, flotillin and TSG101 to characterize the isolated exosomes.

Question 27

Is there any specific biomarker for exosomes isolated from adipocytes?

Accepted Answer

It has been shown that almost all adipocyte-derived exosomes express FABP4.

Question 28

How should I store exosome products?

Accepted Answer

Our exosome products are usually in the form of lyophilized powder or frozen liquid. Lyophilized powder store at 4 °C. Frozen liquid store at -20°C to -80°C. Please avoid freeze/thaw cycles and keep them under optimal storage conditions as described in the instructions.

Product Summary
Name	PNExo™ Exosome-Green Tea
	Download Datasheet
Cat No.	PNE-FLG70
Source	Exosome derived from Green Tea
Product Overview	Plant exosomes are nanosized (30-150 nm) membrane vesicles that contain biomolecules. Plant-derived exosomes refer to naturally occurring nanoparticles derived from plants that contain bioactive molecules and proteins. These exosomes have been shown to have multiple benefits in a variety of applications, such as skincare, drug delivery, and biomedicine. Plant-derived exosomes have been found to possess antioxidant, anti-inflammatory, and anti-aging properties, making them an attractive option for the development of new and innovative therapies. Plant-derived natural substances are widely used as cosmeceutical materials because they exert beneficial effects on the human skin, such as antiaging, moisturizing, whitening, regeneration, and nutritional supply. Besides, they could delivery therapeutic compounds to target cells, potentially revolutionizing the way in which drugs are administered. Overall, plant-derived exosomes hold great promise for a wide range of applications in the fields of medicine and biotechnology. PNExo™ is focused on the production and delivery of high quality plant-derived exosomes products. Exosomes are important tools of intercellular communication with a variety of biological functions, including cell regeneration and immune regulation. PNExo™ products undergo a rigorous screening and purification process that guarantees their high purity and activity. Lyophilization is useful for a long-term storage at 4°C, and frozen liquid should be kept at -20°C to -80°C. Ultracentrifugation and precipitation techniques are mainly used in exosome Isolation. It had been reported that both methods yielded extracellular vesicles in the size range of exosomes and included apoproteins, which can be used in downstream analyses. Creative Biostructure PNExo™ exosome products guarantee higher purity and quality to meet our customer research.
Form	Lyophilized powder
Concentration	> 1x10^6 particles
Storage	Lyophilized powder store at 4 °C. Frozen liquid store at -20°C to -80°C. Recommended to avoid repeated freeze-and-thaw cycles.
Reconstitution	Reconstitute lyophilized exosome by adding deionized water for a desired final concentration. Centrifuge before opening to ensure exosomes are at bottom, resuspend exosomes by pipetting and/or vortex, please avoid bubbles. Centrifuge again and mix well for using.

PNExo™ Exosome-Green Tea(PNE-FLG70)