PureExo® Exosome Isolation kit (for cells)

3 What’s the minimum volume of serum sample can be processed by your PureExo kit (Cat.#: P101)?
A: 50 ul.

4 Which kit should I use for my urine and plasma samples?
A: Use DiagExo® kit (Cat.#: P120) to isolate exosome from urine and Use PureExo® kit for serum (Cat.#: P101) to isolate exosome from serum.

5, Can I use your kit to isolate exosome or endothelial microparticles from cultured cells and from plasma?
A: Our P100 kit can be used to isolate exosome from either cell culture medium or serum. It is not for cells.

6 Is your exosome prep contaminated with IgG and/or beads?
A: We use specific reagents to capture exosomes. We did not use antibody or beads for the isolation. So the final exosome prep is not contaminated with artificial IgG or beads.

7, Do your exosome prep contains Albumin?
A: Some types of cells such as hepatocytes carries enriched Albumin, and these liver derived exosomes can be released into the circulation system. So, if the exosomes from these kinds of samples are isolated, it may contains Albumin.

8, Can I use your PureExo kit (Cat. #P100) on our D.discoideum amoeba?
A: PureExo exosome isolation kit captures released exosomes or microvesicles from cells. For amoeba exosome isolation, we suggest to collect amoeba culture medium as the same way to collect cell culture medium.

9, What is the composition of the buffer that the final exosomes prep will be in? Will it impact my downstream assay?
A: The final isolated exosomes are aggregated nano-particles mixed with a very small amount of original culture medium or serum. PureExo Exosome Isolation kit provides intact exosomes which allows for a varieties of downstream applications including exosome surface labeling10， How do you know that an exosome prep is pure, free of other membrane-derived microparticles
(shredding vesicles)?
A: The isolated microvesicle from cell culture medium or serum using our isolation kits showed sphere membrane encapsulated particles with the diameters varying between 20-200nm under EM scanning. These characters help to determine that the harvested microvesicle are exosomes. Shedding particles are reported to be irregular shape with diameter up to 1000nm. In addition, the size distribution pattern is confirmed by dynamic light scattering indicating the isolated microvesicles are of diameter between 100 and 250nm.

11， Are exosome functionally active?
A: Our collaborators used our kits to detect specific mirRNAs carried by exosomes. Luciferase activity assay
showed active function of exosomal microRNA after the exosomes were applied to cells transfected with targeting mRNA 3’UTR vector.

12， Is it necessary to use glass tubes?
A: Using glass tubes instead of plastic tubes is required in step 3 and step 4.
Please find glass tubes from FisherScientific. http://www.fishersci.com (Search for Fisherbrand* Disposable
Borosilicate Glass Tubes Cat. NO: 14-961-26).

13， Why can’t I see clear fluff middle layer?
A: The following handling errors may lead to the failure:
1). Solution A/B/C did not mix completely before adding the cell culture medium or serum.
2). Did not perform a low speed centrifugation to remove cells and cell debris.
3). The ratio is off. For example: 0.125 mL A + 0.125 mL B + 0.5mL C to 2 mL medium. Overproportion of serum/medium volume to Solution mixture (A/B/C) will lead to an indistinctive interphase.
4). The maximum starting volume is 4 ml medium, and it should be from half million cultured cells or less. If cell number or medium volume exceed this amount, the sample should be splits into multiple samples to proceed.
5). Cultured cells were not starve for 48 hours to remove contaminated exosomes derived from FBS.

14， What biomarker(s) do you use to identify / characterize the isolated exosomes?
A: We use exosome markers such as flotillin, CD9, CD63, Alix, and TSG101 etc to characterize the isolated exosomes.

15， What is the yield of exosome using your kits?
A: For P100 kit (PureExo kit for cell culture medium), typically, 50 ~ 100 ul exosomes can be isolated from 2ml cell culture media (Up to 4 ml of medium can be processed per reaction). If all the harvested exosomes are used for exosomal protein extraction (using our P200 kit), the total protein yield ranges from 150 ~ 200 ug / 2mL medium, 300 ~ 400 ug / 4 ml medium. If all the isolated exosome are used for RNA extraction (using our P200 kit), total exosomal RNA yield is 50 ~ 100 ng / 2ml medium, 100 ~ 200 ng / 4ml medium. For P101 kit (PureExo kit for serum or plasma), typically 100 ~ 200 ul exosome can be isolated from 100ul to 600ul serum. If all the harvested exosomes are used for exosomal protein extraction (using our P200 kit), the total exosomal protein yield ranges from 300-400 ug protein. If all the isolated exosome are used for RNA extraction (using our P200 kit), total exosomal RNA yield is 100 ~ 300ng exosomal RNA For P120 kit (DiagExo kit for Urine), typically 50 ~ 200 ul exosome can be isolated from 3 mL urine. If all the harvested exosomes are used for exosomal protein extraction (using our P200 kit), the total exosomal protein yield ranges from 150-400ug protein. If all the isolated exosome are used for RNA extraction (using our P200 kit), total exosomal RNA yield is 50 ~ 200ng exosomal RNA

16, Can I use heparin or EDTA tube to collect blood sample if I need to isolate exosome from plasma?
A: No. Heparin and EDTA will significantly impair the downstream assays. Use No-heparin-No-EDTA tube to collect the blood sample. Centrifuge the sample immediately to collect the plasma for exosome isolation. .