CN111961636B - Exosome extraction reagent and application thereof - Google Patents
Exosome extraction reagent and application thereof Download PDFInfo
- Publication number
- CN111961636B CN111961636B CN202010642250.0A CN202010642250A CN111961636B CN 111961636 B CN111961636 B CN 111961636B CN 202010642250 A CN202010642250 A CN 202010642250A CN 111961636 B CN111961636 B CN 111961636B
- Authority
- CN
- China
- Prior art keywords
- exosome
- reagent
- hela
- cell culture
- iii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses an exosome extraction reagent, which comprises 10-200mM of PEG4000, 10-50mM of beta-cyclodextrin, 10-50mM of imidazole, 10-50mM of copper sulfate and 10-50mM of HEPES. The invention also discloses application of the reagent in separating cell culture fluid exosomes. Compared with an Invitrogen kit method and an SBI kit method, the reagent provided by the invention has the advantages that the number of extracted exosomes is more, the particle size distribution is more concentrated, and the morphology background of an electron microscope is clearer. Meanwhile, the exosome preparation reagent provided by the invention is simple to operate, low in cost, high in yield and high in purity, and can be used for exosome protein, nucleic acid, electron microscope and particle lens analysis.
Description
Technical Field
The invention relates to the field of biomedicine, and particularly belongs to a reagent and a method for separating a cell culture solution exosome.
Background
The exosome is a vesicle with a diameter of 30-150nm and wrapped by double-layer lipid molecules, is selectively packaged and released by living cells, and is rich in human body fluid. The exosome contains various lipids, nucleic acids, proteins and the like, and the substances can be transported to a specific target organ and related target cells along with body fluid so as to exert corresponding biological functions. Related studies have shown that exosomes play a tremendous role in cell-cell communication as well as in physiological and pathological processes.
In recent years, as the research and the recognition of exosomes are deepened, exosome detection is widely applied to noninvasive diagnosis, treatment and monitoring of tumors and diseases by a plurality of clinical scientific research institutions as a novel liquid biopsy hotspot technology; how to extract exosomes efficiently is the key to realizing the clinical routine application of the emerging liquid biopsy technology.
The standard of exosome extraction at present is still ultra-high-speed centrifugation. The typical disadvantages of ultracentrifugation are that the operation is complicated, the required equipment is expensive, and it is difficult to obtain sufficient amount of exosome for subsequent experimental analysis. Meanwhile, the equipment requirement of the ultracentrifuge and the training of the operation program greatly improve the extraction cost, and the clinical routine application cannot be realized. Because of the shortage of exosome extraction reagents in China, the research on exosomes in China basically depends on the ultracentrifugation and import extraction kit with complicated processes, so the development of the research and clinical application of exosomes in China is seriously hindered. Therefore, there is a need for a method for obtaining high-purity exosomes easily and conveniently.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the defects of the prior art, the invention provides the extraction reagent which has low cost and convenient operation and is suitable for extracting high-purity exosome in cell culture solution and the separation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows: an exosome-extracting reagent comprising 10-200mM PEG4000, 10-50mM beta-cyclodextrin, 10-50mM imidazole, 10-50mM copper sulfate and 10-50mM HEPES.
Preferably, the reagent comprises 100-200mM PEG4000, 20-50mM beta-cyclodextrin, 10-20mM imidazole, 20-50mM copper sulfate and 20-50mM HEPES.
In a preferred embodiment, the reagent comprises 100mM PEG4000, 40mM β -cyclodextrin, 10mM imidazole, 20mM copper sulfate and 20mM HEPES.
The invention further provides application of the reagent in separation of cell culture fluid exosomes.
Specifically, the application process comprises the following steps:
(1) firstly, centrifuging cell culture solution, and taking supernatant;
(2) uniformly mixing the reagent of claim 1 with the supernatant obtained in the step (1), and standing overnight; the next day, centrifugation is carried out again, the supernatant is discarded, and the obtained precipitate is the high-purity exosome.
Wherein, in the step (1), the centrifugation condition is centrifugation for 10-20min at 4 ℃ and under the condition of 3000-.
Preferably, in step (2), the ratio of the reagent: the volume ratio of the supernatant is 1: 1-1: 10.
further preferably, in the step (2), the centrifugation is performed at 4 ℃ and 5000-.
In a preferred embodiment, the exosomes are isolated using the following technical scheme: firstly, centrifuging the cell culture solution for 10min at 4 ℃ under 3000 g; and mixing the reagents according to the proportion of 1: 1-1: 10 (reagent: cell culture solution) in a volume ratio, and uniformly mixing; 4 ℃, standing overnight: centrifuging for 60min at 4 deg.C and 5000g the next day, and discarding supernatant to obtain precipitate as high-purity exosome.
Has the advantages that: compared with an Invitrogen kit method and an SBI kit method, the reagent provided by the invention has the advantages that the number of extracted exosomes is more, the particle size distribution is more concentrated, and the morphology background of an electron microscope is clearer. Meanwhile, the exosome preparation reagent provided by the invention is simple to operate, low in cost, high in yield and high in purity, and can be used for exosome protein, nucleic acid, electron microscope and particle lens analysis.
Drawings
FIG. 1 shows the results of detection of exosome markers HSP70 and CD63 proteins by Western Blot after exosomes were prepared from different cell culture fluid samples using the reagent of the present invention;
FIG. 2 shows the results of particle size (NTA) detection of exosomes prepared using the reagent of the present invention in different cell culture fluid samples;
FIG. 3 shows the results of electron microscope imaging tests performed on exosomes prepared by using the reagent of the present invention in different cell culture fluid samples;
FIG. 4 shows the detection results of the exosome marker proteins HSP70 and CD63 by Western Blot after exosomes are prepared by the reagent and Invitrogen kit method and the SBI kit method;
FIG. 5 shows the results of particle size (NTA) detection performed after exosome preparation using the reagent of the present invention and Invitrogen kit method and SBI kit method;
FIG. 6 shows the results of electron microscopy imaging detection performed after exosomes were prepared with the reagent of the present invention, the Invitrogen kit method, and the SBI kit method.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The detailed embodiments and specific procedures are given, and the types of cell culture fluids and the combination of reagents listed in the examples will help to understand the present invention, but the scope of the present invention is not limited to the following examples, and the types of cell culture fluids are not limited in practical applications.
The materials, equipment and methods adopted by the invention are conventional materials, equipment and methods in the technical field, wherein PEG4000, beta-cyclodextrin, imidazole, copper sulfate and HEPES are purchased from chemical reagents of national medicine group, Inc.; Anti-Hsp70 antibody [ EPR16892] (ab181606) and Anti-CD63 antibody-Late Endosome Marker (ab216130) were purchased from Abcam corporation. The materials and reagents used in the following examples are commercially available unless otherwise specified.
Example 1 the cell culture solutions of a549, K562, HepG2, HeLa were subjected to exosome extraction using PEG4000 solutions of different concentrations, respectively.
1) Preparing PEG4000 solutions with the concentrations of 10mM, 100mM and 200mM respectively, wherein the numbers are I, II and III in sequence;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated. Then adding 250 mu L of PEG4000 solution (not added in the untreated group) according to the corresponding number respectively, and uniformly mixing; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 1.
TABLE 1
From the experimental results, it is found that when the concentration of PEG4000 is 100mM or less, the higher the concentration, the larger the amount of exosome extracted from the cell culture solution, but when the concentration of PEG4000 is 100mM or more, the higher the concentration, the less significant the increase of the amount of exosome extracted from the cell culture solution.
Example 2 cell culture solutions of a549, K562, HepG2, and HeLa were subjected to exosome extraction using β -cyclodextrin solutions of different concentrations, respectively.
1) Preparing beta-cyclodextrin solutions with the concentrations of 10mM, 20mM and 40mM respectively, wherein the serial numbers are I, II and III in sequence;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated. Then adding 250 mu L of beta-cyclodextrin solution (not added in the untreated group) according to the corresponding number respectively, and uniformly mixing; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 2.
TABLE 2
From the results of the experiments, it was found that, when the concentration of β -cyclodextrin was 40mM or less, the higher the concentration was, the larger the amount of exosomes extracted from the cell culture solution was.
Example 3 cell culture solutions of a549, K562, HepG2, and HeLa were subjected to exosome extraction using imidazole solutions of different concentrations, respectively.
1) Preparing imidazole solutions with the concentrations of 10mM, 30mM and 50mM respectively, wherein the numbers of the imidazole solutions are I, II and III in sequence;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated. Then adding 250 mu L of imidazole solution (not added in the untreated group) according to the number respectively, and uniformly mixing; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 3.
TABLE 3
According to the experimental results, the imidazole basically has no influence on the extraction of exosomes in the cell culture solution.
Example 4 the cell culture solutions of a549, K562, HepG2, HeLa were subjected to exosome extraction using copper sulfate solutions of different concentrations, respectively.
1) Preparing copper sulfate solutions with the concentrations of 10mM, 20mM and 40mM respectively, wherein the serial numbers are I, II and III in sequence;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated. Then adding 250 mu L of copper sulfate solution (not added in the untreated group) according to the corresponding number respectively, and uniformly mixing; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 4.
TABLE 4
4) According to the experimental results, copper sulfate has no influence on the extraction of exosomes in the cell culture solution.
Example 5 cell culture solutions of a549, K562, HepG2, HeLa were subjected to exosome extraction using HEPES solutions of different concentrations, respectively.
1) Preparing HEPES solutions with the concentrations of 10mM, 20mM and 40mM respectively, wherein the serial numbers are I, II and III in sequence;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated. Then adding 250 mu L of HEPES solution (not added in the untreated group) according to the corresponding number respectively, and uniformly mixing; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 5.
TABLE 5
According to the experimental results, the HEPES has no influence on the extraction of exosomes in the cell culture solution.
Example 6 exosome extractions were performed on cell culture solutions of a549, K562, HepG2, HeLa, respectively, using different combinations of reagents.
1) Preparing reagents of different combinations: 100mM PEG4000, 20mM copper sulfate and 20mM HEPES, which are marked as reagent I; beta-cyclodextrin of 40mM, copper sulfate of 20mM and HEPES of 20mM, and is marked as reagent II; imidazole of 10mM, copper sulfate of 20mM and HEPES of 20mM, which are marked as reagent III; 100mM PEG4000, 40mM beta-cyclodextrin, 20mM copper sulfate and 20mM HEPES, which are marked as reagent IV; 100mM PEG4000, 10mM imidazole, 20mM copper sulfate and 20mM HEPES, which are marked as reagent V; beta-cyclodextrin of 40mM, imidazole of 10mM, copper sulfate of 20mM and HEPES of 20mM, and is marked as a reagent VI;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 7 centrifuge tubes, 1mL of each cell is obtained, and the total of 4 cells needs 28 centrifuge tubes. The serial numbers of the components are A549I, A549 II, A549 III, A549 IV, A549V, A549 VI and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 IV, K562V, K562 VI, K562 No; HepG 2I, HepG2 II, HepG2 III, HepG2 IV, HepG 2V, HepG2 VI and HepG2 are not treated; HeLa I, HeLa II, HeLa III, HeLa IV, HeLa V, HeLa VI, HeLa untreated. Then adding 250 mu L of reagent (not added in the untreated group) according to the number respectively, and mixing uniformly; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection result is shown in Table 6.
TABLE 6
The results of examples 1-6 show that PEG4000 has a certain effect of adsorbing exosomes, beta-cyclodextrin has a slight effect of adsorbing exosomes, and imidazole, copper sulfate and HEPES are used alone and have almost no effect of adsorbing exosomes. However, when several substances of PEG4000, beta-cyclodextrin, imidazole, copper sulfate and HEPES are used together, obvious adsorption effect on exosome can be generated.
Example 7 exosome extraction was performed on cell culture fluids of a549, K562, HepG2, HeLa, respectively, using the reagents of the present invention at different concentration ratios.
1) Preparing reagents with different concentration ratios: 10mM PEG4000, 20mM beta-cyclodextrin, 10mM imidazole, 10mM copper sulfate and 10mM HEPES, which are marked as reagent I; 50mM PEG4000, 40mM beta-cyclodextrin, 10mM imidazole, 20mM copper sulfate and 10mM HEPES, which are marked as reagent II; 100mM PEG4000, 40mM beta-cyclodextrin, 10mM imidazole, 20mM copper sulfate and 20mM HEPES, which are marked as reagent III; 100mM PEG4000, 40mM beta-cyclodextrin, 20mM imidazole, 20mM copper sulfate and 20mM HEPES, which are marked as reagent IV; 100mM PEG4000, 50mM beta-cyclodextrin, 20mM imidazole, 30mM copper sulfate and 30mM HEPES, which is marked as reagent V; 200mM PEG4000, 50mM beta-cyclodextrin, 50mM imidazole, 50mM copper sulfate and 50mM HEPES, which are marked as a reagent VI;
2) cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 7 centrifuge tubes, 1mL of each cell is obtained, and the total of 4 cells needs 28 centrifuge tubes. The serial numbers of the components are A549I, A549 II, A549 III, A549 IV, A549V, A549 VI and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 IV, K562V, K562 VI, K562 No; HepG 2I, HepG2 II, HepG2 III, HepG2 IV, HepG 2V, HepG2 VI and HepG2 are not treated; HeLa I, HeLa II, HeLa III, HeLa IV, HeLa V, HeLa VI, HeLa untreated. Then adding 250 mu L of reagent (not added in the untreated group) according to the number respectively, and mixing uniformly; standing the mixed solution for 12h at 4 ℃; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
3) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection results are shown in Table 7.
TABLE 7
4) And (3) extracting the obtained exosome heavy suspensions by using a reagent III, adding equal volume of RIPA lysate to perform lysis and protein extraction, and performing exosome marker HSP70 and CD63 protein detection by Western Blot, wherein the detection result is shown in figure 1.
5) The exosome weight suspension obtained by extracting reagent III is selected, 20 mu L of the exosome weight suspension is diluted by 100 times respectively, and then the particle size (NTA) detection is carried out, and the detection results are shown in Table 8 and figure 2.
TABLE 8
| Numbering | A549Ⅲ | K562Ⅲ | HepG2Ⅲ | HeLaⅢ |
| Average particle diameter (nm) | 94.2 | 122.7 | 87.9 | 106.9 |
| Particle concentration (one/mL) | 4.55×1011 | 6.62×109 | 5.13×1012 | 3.29×1012 |
6) Selecting a reagent III to extract the obtained exosome heavy suspensions, taking 10 mu L of exosome heavy suspensions respectively, carrying out related detection on a transmission electron microscope, adopting a phosphotungstic acid negative staining method, and specifically comprising the following steps: sucking 10 mu L of sample, dripping the sample on a copper net for 1min, sucking the floating liquid by using filter paper, dripping 10 mu L of 2 percent phosphotungstic acid on the copper net for 1min, sucking the floating liquid by using the filter paper, then putting the copper net on the filter paper, drying the copper net for a plurality of minutes at normal temperature, and carrying out electron microscope detection imaging at 100KV, wherein the detection result is shown in figure 3.
The experimental results show that the reagent III combination is preferably selected in the invention: 100mM PEG4000, 40mM beta-cyclodextrin, 10mM imidazole, 20mM copper sulfate and 20mM HEPES. The reagent and the related experimental method provided by the invention are suitable for extracting exosomes from different cell culture fluid samples, the operation is simple, and the purity of the obtained exosomes is high.
EXAMPLE 8 comparison of the inventive reagents with Invitrogen and SBI reagents for exosome concentration and purity in cell culture broth extracts
1) Cell culture fluid samples of A549, K562, HepG2 and HeLa are respectively taken, centrifuged for 10min at 4 ℃ and 3000g, supernatant is taken, each cell is respectively filled in 4 centrifuge tubes, 1mL of each cell is obtained, and 16 centrifuge tubes are needed in total for 4 cells. The serial numbers of the components are A549I, A549 II, A549 III and A549 without treatment in sequence; K562I, K562 II, K562 III, K562 were untreated; HepG 2I, HepG2 II, HepG2 III and HepG2 were untreated; HeLa I, HeLa II, HeLa III, HeLa untreated.
2) Group I cell culture fluid was extracted according to the combination of reagents III of the present invention in example 7, the extraction procedure was as described in example 7, and the resulting exosome pellet was resuspended in 100. mu.L PBS;
3) operating the group II cell culture solution according to the standard flow of the Invitrogen kit, adding 1mL of extraction reagent into the cell culture solution, and uniformly mixing; standing for 12h at 4 ℃; centrifugation was carried out at 4 ℃ and 10000g for 60min, the supernatant was discarded, and the resulting exosome pellet was resuspended in 100. mu.L PBS.
4) Operating the group III cell culture solution according to the standard process of the SBI kit, adding 200 mu L of extraction reagent into the cell culture solution, and uniformly mixing; standing at room temperature for 60 min; standing for 12h at 4 ℃; centrifugation at 1500g for 30min at 4 ℃ was carried out, the supernatant was discarded and the resulting exosome pellet was resuspended in 100. mu.L LPBS.
5) Adding no extraction reagent into the untreated group, and standing at 4 deg.C for 12 h; centrifuging at 4 deg.C and 5000g for 60 min; the supernatant was discarded and the resulting exosome pellet was resuspended in 100 μ L PBS.
6) 10 mu L of each obtained exosome suspension is taken, an equal volume of RIPA lysate is added for lysis, the content of the obtained exosome protein is detected by a BCA method, and the detection results are shown in Table 9.
TABLE 9
7) Three methods are selected to extract exosome heavy suspensions obtained by K562 cell culture solution, 20 mu L of exosome heavy suspensions are respectively added with equal volume of RIPA lysate for lysis and protein extraction, and exosome marker proteins HSP70 and CD63 are detected by Western Blot, and the detection result is shown in figure 4.
8) Three methods are selected to extract the exosome weight suspension obtained by K562 cell culture solution, 20 mu L of exosome weight suspension is diluted by 100 times respectively, and then particle size (NTA) detection is carried out, and the detection results are shown in table 10 and figure 5.
| Numbering | K562Ⅰ | K562Ⅱ | K562Ⅲ |
| Average particle diameter (nm) | 99.0 | 143.0 | 146.5 |
| Particle concentration (one/mL) | 4.18×1012 | 6.06×1011 | 7.62×108 |
9) Three methods are selected to extract exosome heavy suspensions obtained by K562 cell culture solution, 10 mu L of exosome heavy suspensions are respectively taken to carry out related detection of a transmission electron microscope, a phosphotungstic acid negative staining method is adopted, and the method specifically comprises the following steps: sucking 10 mu L of sample, dripping the sample on a copper net for 1min, sucking the floating liquid by using filter paper, dripping 10 mu L of 2% phosphotungstic acid on the copper net for 1min, sucking the floating liquid by using the filter paper, then putting the copper net on the filter paper, drying the copper net for a plurality of minutes at normal temperature, and carrying out electron microscope detection imaging at 100KV, wherein the detection result is shown in figure 6.
Compared with an Invitrogen kit method and an SBI kit method, the content of the exosome marker in the cell culture solution is higher, and the obtained particle number is the largest.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. An exosome extraction reagent, which is characterized in that the reagent comprises 10-200mM of PEG4000, 10-50mM of beta-cyclodextrin, 10-50mM of imidazole, 10-50mM of copper sulfate and 10-50mM of HEPES.
2. The reagent as claimed in claim 1, wherein the reagent comprises 100-200mM PEG4000, 20-50mM β -cyclodextrin, 10-20mM imidazole, 20-50mM copper sulfate and 20-50mM HEPES.
3. The reagent of claim 1, wherein the reagent comprises 100mM PEG4000, 40mM β -cyclodextrin, 10mM imidazole, 20mM copper sulfate and 20mM HEPES.
4. Use of the agent of claim 1 for the isolation of cell culture fluid exosomes.
5. Use according to claim 4, characterized in that it comprises the following steps:
(1) firstly, centrifuging cell culture solution, and taking supernatant;
(2) uniformly mixing the reagent of claim 1 with the supernatant obtained in the step (1), and standing overnight; the next day, centrifugation is carried out again, the supernatant is discarded, and the obtained precipitate is the high-purity exosome.
6. The use as claimed in claim 5, wherein in step (1), the centrifugation is carried out at 4 ℃ and 3000-5000g for 10-20 min.
7. Use according to claim 5, wherein in step (2) the ratio of reagent: the volume ratio of the supernatant is 1: 1-1: 10.
8. the use according to claim 5, wherein in step (2), the centrifugation is performed at 4 ℃ and 5000-10000g for 30-60 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010642250.0A CN111961636B (en) | 2020-07-06 | 2020-07-06 | Exosome extraction reagent and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010642250.0A CN111961636B (en) | 2020-07-06 | 2020-07-06 | Exosome extraction reagent and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111961636A CN111961636A (en) | 2020-11-20 |
| CN111961636B true CN111961636B (en) | 2021-11-26 |
Family
ID=73361083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010642250.0A Active CN111961636B (en) | 2020-07-06 | 2020-07-06 | Exosome extraction reagent and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111961636B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112813027B (en) * | 2020-12-31 | 2023-05-05 | 南方科技大学 | Method for separating exosomes in urine |
| CN117187165A (en) * | 2023-09-12 | 2023-12-08 | 知行健康产业(广东)有限责任公司 | Method for separating exosomes |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105748437A (en) * | 2014-12-17 | 2016-07-13 | 北京盈科瑞药物研究院有限公司 | Vesicle and vesicle preparation, and preparation methods thereof |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN107893050A (en) * | 2017-10-17 | 2018-04-10 | 杜水果 | A kind of extracellular vesica and its production and use |
| WO2018131966A1 (en) * | 2017-01-13 | 2018-07-19 | (주)로제타엑소좀 | Extracellular vesicle isolation method using metal |
| CN108795938A (en) * | 2018-06-21 | 2018-11-13 | 中国科学院北京基因组研究所 | The special miRNA of adenocarcinoma of lung excretion body and its target gene and application |
| WO2019079743A1 (en) * | 2017-10-19 | 2019-04-25 | Streck, Inc. | Compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles |
| CN110177544A (en) * | 2016-11-29 | 2019-08-27 | 普尔泰克健康有限公司 | For delivering the excretion body of therapeutic agent |
| CN110643032A (en) * | 2016-04-21 | 2020-01-03 | 厦门赛诺邦格生物科技股份有限公司 | Eight-arm polyethylene glycol, preparation method, functionalized derivative and modified biologically-relevant substance |
| CN111148828A (en) * | 2017-07-26 | 2020-05-12 | 罗塞塔外排体株式会社 | Method for separating extracellular vesicles using cations |
| CN112322612A (en) * | 2020-10-29 | 2021-02-05 | 江苏凯基生物技术股份有限公司 | Plasmid extraction kit and extraction method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4003305A4 (en) * | 2019-07-22 | 2023-07-26 | Erivan Bio, LLC | Topical exosome compositions and associated methods |
-
2020
- 2020-07-06 CN CN202010642250.0A patent/CN111961636B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105748437A (en) * | 2014-12-17 | 2016-07-13 | 北京盈科瑞药物研究院有限公司 | Vesicle and vesicle preparation, and preparation methods thereof |
| CN110643032A (en) * | 2016-04-21 | 2020-01-03 | 厦门赛诺邦格生物科技股份有限公司 | Eight-arm polyethylene glycol, preparation method, functionalized derivative and modified biologically-relevant substance |
| CN110177544A (en) * | 2016-11-29 | 2019-08-27 | 普尔泰克健康有限公司 | For delivering the excretion body of therapeutic agent |
| WO2018131966A1 (en) * | 2017-01-13 | 2018-07-19 | (주)로제타엑소좀 | Extracellular vesicle isolation method using metal |
| CN111148828A (en) * | 2017-07-26 | 2020-05-12 | 罗塞塔外排体株式会社 | Method for separating extracellular vesicles using cations |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN107893050A (en) * | 2017-10-17 | 2018-04-10 | 杜水果 | A kind of extracellular vesica and its production and use |
| WO2019079743A1 (en) * | 2017-10-19 | 2019-04-25 | Streck, Inc. | Compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles |
| CN108795938A (en) * | 2018-06-21 | 2018-11-13 | 中国科学院北京基因组研究所 | The special miRNA of adenocarcinoma of lung excretion body and its target gene and application |
| CN112322612A (en) * | 2020-10-29 | 2021-02-05 | 江苏凯基生物技术股份有限公司 | Plasmid extraction kit and extraction method |
Non-Patent Citations (4)
| Title |
|---|
| Immuno-modified superparamagnetic nanoparticles via host–guest interactions for high-purity capture and mild release of exosomes;Shuang Cai等;《Nanoscale》;20180807;第10卷(第29期);第1-10页 * |
| Isolation of Extracellular Vesicles from Stem Cells;Chen Zixin等;《Methods Mol Biol.》;20170913;第389-394页 * |
| 外泌体分离与鉴定方法的研究进展;龚春梅等;《生命科学》;20180326;第30卷(第3期);第319-326页 * |
| 外泌体提取方法及鉴定分析研究进展;贺娇等;《中华实用诊断与治疗杂志》;20180711;第32卷(第7期);第718-721页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111961636A (en) | 2020-11-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111961636B (en) | Exosome extraction reagent and application thereof | |
| CN109564147B (en) | Systems and methods for analyzing analytes extracted from a sample using an adsorbent material | |
| CN108220125A (en) | A kind of nucleic acid rapid extraction device | |
| CN110511902A (en) | Based on the separation of the extracellular vesica of exclusion chromatography and hyperfiltration technique and enrichment method | |
| CN110951669A (en) | Coprecipitator, reagent group, kit and extraction method for extracting exosome | |
| CN113774008A (en) | Method for extracting exosome and application thereof | |
| CN108865978A (en) | A method of separation and purifying excretion body | |
| CN107525818A (en) | A kind of method and reagent that excretion body is extracted from urine | |
| CN109609458A (en) | A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body | |
| CN108801746A (en) | A kind of device of separation and enrichment body fluid components | |
| CN109266613A (en) | A kind of excretion body method for separating and preparing | |
| CN112852725B (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN209485831U (en) | The extracellular vesica enriching apparatus of the urine of hospital | |
| CN110747255A (en) | Whole blood sample pretreatment reagent and method for nucleic acid detection | |
| CN113101737A (en) | Affinity tangential flow filtration system and construction method thereof, and exosome extraction method and application | |
| CN112094809A (en) | Method for extracting exosome from serum or plasma | |
| CN110068618B (en) | Detection method of intestinal flora metabolites related to nephropathy | |
| CN103900890A (en) | Method for extracting urinary micro vesicle using nanofilm concentration | |
| CN110438061A (en) | A method of separating excretion body from fluid shear stress perfusate | |
| CN114397388B (en) | Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application | |
| CN209485829U (en) | The device of the workable enrichment urine excretion body of hospital and family | |
| CN113913367A (en) | Exosome separation and enrichment method based on exosome enrichment device | |
| CN112980006A (en) | Protein cross-linked nano affinity microsphere, preparation method and application | |
| CN209296429U (en) | The urine excretion body enrichment system that suitable basic hospital uses | |
| CN116555160B (en) | Extraction method of radix paeoniae alba exosome and decoction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |