A method for extracting extracellular vesicles from body fluids based on a two-phase aqueous system
A two-phase, vesicle technology, which is used in biochemical equipment and methods, preparation of test samples, and determination/inspection of microorganisms, can solve the problems of complex operation, low recovery rate, long time, etc. Quick and easy effects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
experiment example 1
[0043]Experimental example 1 ATPS phase separation curve
[0044]1. Reagent preparation
[0045]The polymer polyethylene glycol (PEG) has a molecular weight of 35,000 and dextran (DEX) has a molecular weight of 450,000-650,000, which are respectively diluted in PBS to prepare a stock solution with a concentration of 35% wt and 15% wt. Mix 35% wt PEG and PBS to prepare a solution with a total volume of 1 mL, and the PEG concentration is 2%, 4%, 6%, 8%, and 10%. PBS buffer solution with pH 7.4.
[0046]2. Determination of ATPS phase transition curve
[0047]Then add 15%wt DEX solution dropwise to it until the mixed solution appears turbid, record the volume of DEX solution used, and convert it to the final PEG and DEX concentration to get the phase diagram.figure 2 (The abscissa represents the DEX concentration, the ordinate represents the PEG concentration, and the ATPS phasegraph represents the ATPS phase diagram). The results show that when the concentration of PEG and DEX is above the curve, ...
experiment example 2A
[0048]Experiment 2 Optimization of ATPS polymer concentration
[0049]1. Reagent preparation
[0050]Use polymer polyethylene glycol (PEG) molecular weight 35,000, dextran (DEX) molecular weight 450,000-650,000. Prepare PEG 35,000 solutions with concentrations of 60%, 50%, and 40%, and DEX 450,000-650,000 solutions with concentrations of 10%, 15%, and 20% respectively. Cell membrane green fluorescent probe DIO. PBS buffer solution with pH 7.4.
[0051]2. Preparation of green fluorescently labeled extracellular vesicles
[0052]The commercial human plasma was thawed and centrifuged at 1300g for 15 minutes to remove cell debris. Then transfer the supernatant to a new centrifuge tube, centrifuge at 3000g for 15 minutes, mix the supernatant with PBS in a volume ratio of 1:9, and centrifuge at 13000g for 30 minutes at 4°C. Dispense the supernatant into 20mL ultra-isolation tubes (2mL plasma per tube), centrifuge at 100,000g for 1 hour, discard the supernatant, resuspend the pellet at the bottom of t...
experiment example 3
[0058]Experimental Example 3 The effect of plasma dilution on the extraction of extracellular vesicles
[0059]1. Reagent preparation
[0060]Use polymer polyethylene glycol (PEG) molecular weight 35,000, dextran (DEX) molecular weight 450,000-650,000. Prepare a PEG 35,000 solution with a concentration of 50% and a DEX solution with a concentration of 15%. PBS buffer solution with pH 7.4.
[0061]2. Preparation of green fluorescently labeled extracellular vesicles
[0062]Same as Experimental Example 2
[0063]3. Preparation of human plasma without extracellular vesicles
[0064]Same as Experimental Example 2
[0065]4. Optimization of plasma dilution
[0066]Add 1 mL of green fluorescently labeled extracellular vesicles to 9 mL of human plasma without vesicles to form standard A. Dilute standard product A and PBS with 1:1 (standard product B), 1:2 (standard product C) and 1:3 (standard product D), and then mix standard products A, B, C and D, respectively Take 800μL mixed with 100μL 50% PEG and 100μL 15% ...
PUM
| Property | Measurement | Unit |
|---|---|---|
| quality score | aaaaa | aaaaa |
| quality score | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More