CN113046304A - Method for separating mitochondria - Google Patents
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- CN113046304A CN113046304A CN202110383807.8A CN202110383807A CN113046304A CN 113046304 A CN113046304 A CN 113046304A CN 202110383807 A CN202110383807 A CN 202110383807A CN 113046304 A CN113046304 A CN 113046304A
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Abstract
The invention discloses a mitochondrial separation method, which comprises the following steps: 1) re-suspending the cultured cells by using the separation liquid to obtain a cell suspension; 2) the Teflon grinder is connected to the IKA stirrer to grind the cell suspension; 3) centrifuging to obtain a supernatant; 4) centrifuging the supernatant, and collecting precipitate; the separation method has the advantages of low cost, simple operation, time consumption reduction, improvement of the final mitochondrial yield, contribution to obtaining complete mitochondria, contribution to keeping the energy metabolism activity of the mitochondria and carrying out subsequent metabolism-related detection.
Description
Technical Field
The invention relates to the technical field of biotechnology, in particular to a mitochondrial separation method.
Background
Mitochondria are an important organelle in cells, and are important organelles which are unique to eukaryotic cells, exist in most living cells, and are responsible for energy conversion.
The energy metabolism function of mitochondria, namely oxidative phosphorylation, becomes a hot target of current drug development, at present, a plurality of oxidative phosphorylation inhibitors are reported to be applicable to the treatment of various cancers, and part of drugs enter a clinical test stage. The electron transfer chain is the main structure of mitochondria for oxidative phosphorylation, and consists of 5 complexes, and the research on the influence of drugs on the activity of the complexes is an important basis for screening oxidative phosphorylation inhibitors. Current kits for detecting the activity of these complexes require that intact mitochondria be isolated and their energy metabolism activity be maintained as far as possible.
The existing reported method and the commercially available kit have high cost and inconvenient use.
Disclosure of Invention
In view of this, the present application provides a method for separating mitochondria, which has low cost and simple operation, shortens the time consumption, increases the final yield of mitochondria, is beneficial to maintaining the energy metabolism activity of mitochondria, and is beneficial to obtaining complete mitochondria.
Solves the problems of high cost, low yield, slow reaction speed, poor effect and inconvenient use in the prior art.
In order to solve the above technical problems, the present application provides a method for separating mitochondria, comprising:
1) re-suspending the cultured cells by using the separation liquid to obtain a cell suspension; the separating liquid is used for re-suspending and culturing the cells to maintain the energy metabolism activity of the cells;
2) a Teflon grinder connected to the IKA stirrer grinds the cell suspension to break the cell membrane without damaging intracellular organelles;
3) centrifuging to obtain a supernatant;
4) centrifuging the supernatant, and collecting precipitate.
Preferably, the steps 1) to 4) are performed on ice.
Preferably, the method further comprises: the centrifuge was precooled to 4 ℃ before centrifugation.
Preferably, the cultured cells are animal cells.
Preferably, the cultured cells are mammalian cells.
Preferably, the cultured cells are hepatic stellate cells.
Preferably, the cultured cells are rat hepatic stellate cells.
Preferably, the step 1) specifically comprises: for an adherent cell culture system, the culture medium is sucked from the culture system, and the separation liquid is adopted to rinse the cultured cells; collecting cells by adopting a scraping method and re-suspending the cells by adopting the separation liquid to culture the cells to obtain a cell suspension; or
For suspension cell culture systems, cultured cells are directly collected by centrifugation, rinsed with the separation solution, and resuspended with the separation solution.
Preferably, the step 1) specifically comprises:
A) for an adherent cell culture system, placing the culture system containing culture cells and a culture medium in a sample container, sucking and discarding the culture medium by a liquid transfer gun, rinsing by adopting the separating solution, and discarding the separating solution;
B) adding the separation liquid, scraping off the target cells on the wall of the sample container by using a cell scraper, and collecting the cells;
C) adding the separation solution, and collecting the residual cells;
D) combining the cells collected in step B) and the remaining cells collected in step C) to obtain a cell suspension.
Preferably, the addition amount of the separation liquid in the step B) and the step C) is 2mL/75cm2。
Preferably, the sample container is a petri dish or a culture flask.
Preferably, the sample container is a T75 culture flask.
Preferably, the separation liquid contains saccharides, EGTA, HEPES, and serum albumin.
Preferably, the final concentration of the saccharides in the separation liquid is 278-320 mM, the final concentration of EGTA is 0.1-1 mM, the final concentration of HEPES is 5-10 mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the sugar is sucrose and/or mannitol, and the animal serum albumin is bovine serum albumin.
Preferably, the final concentration of sucrose in the separation solution is 320mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the final concentration of sucrose in the separation solution is 70mM, the final concentration of mannitol is 210mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the bovine serum albumin is fatty acid-free bovine serum albumin.
Preferably, the separation medium contains sucrose at a final concentration of 320mM, EGTA at a final concentration of 1mM, HEPES at a final concentration of 10mM, and fatty acid-free bovine serum albumin at a final concentration of 0.5% (g/mL).
Preferably, the pH value of the separation liquid is 7.2.
Preferably, the preparation method of the separation liquid specifically comprises the following steps:
mixing sugar, EGTA, HEPES, animal serum albumin and water;
and adjusting the pH value.
Preferably, the preparation method of the separation liquid specifically comprises the following steps:
mixing sucrose, EGTA, HEPES, fatty acid-free bovine serum albumin and water; the final concentration of sucrose in the separating medium is 320mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5% (g/mL);
the pH was adjusted to 7.2 using KOH.
Preferably, the separation solution is formulated to be stored at 4 ℃ for 1 month and at-20 ℃ for 6 months.
Preferably, the stirring speed of the stirring treatment in the step 2) is 300-500 rpm.
Preferably, the stirring speed of the stirring treatment in the step 2) is 350 revolutions per minute.
Preferably, the number of times of up-and-down grinding is more than or equal to 3, and the cell breakage rate of the cell suspension is more than or equal to 50%.
Preferably, the number of times of up-and-down grinding is more than or equal to 5.
Preferably, the number of times of up-and-down grinding is 10.
Preferably, cell disruption rate is observed using trypan blue staining.
Preferably, in the step 3), the rotating speed is 700-1000 Xg, and the centrifugation is carried out for 5-10 min at the temperature of 0-4 ℃;
and 4) centrifuging at the rotating speed of 3000-6000 Xg in the step 4) for 15min at the temperature of 0-4 ℃.
Preferably, the centrifugation treatment in the step 3) is performed at the rotation speed of 700Xg for 10min at 4 ℃.
Preferably, the centrifugation treatment in the step 4) is performed at the rotating speed of 3000Xg for 15min at the temperature of 4 ℃.
Preferably, the method further comprises: adding separated liquid to resuspend mitochondria for activity detection.
Compared with the prior art, the detailed description of the application is as follows:
the methods and commercially available kits reported in the prior art are mostly used for purifying mitochondria to obtain mitochondrial proteins, DNA and other components, and are focused on purity, i.e. are not polluted by other organelles or cytoplasmic components, and detergents are often used for improving the yield of mitochondria; or gradient centrifugation is used, so that the operation is complicated, the requirement on the proficiency of operators is high, the consumed time is long, the loss of energy metabolism activity of mitochondria is easily caused, and the requirement on centrifugal equipment is also high. The separation method specially aiming at the mitochondrial activity test generally adopts a DOUNCE grinder to grind manually, the grinding times are few, the cell breakage rate is low, a large amount of cells are needed, the grinding times are many, although the cell breakage rate is increased, the time and the labor are wasted, and the method is not beneficial to simultaneously processing a plurality of samples.
That is, the separation of the intact mitochondria with metabolic activity from the cell is a precondition for detecting the activity of the electron transport chain complex, some prior art contain detergent, which affects the energy metabolic activity of mitochondria, some have many steps, complicated operation, long time consumption, which is not good for the maintenance of the energy metabolic activity of mitochondria, and some have high requirements for the centrifugal equipment.
1. The separation method only collects cells, grinds, centrifuges and centrifuges, and has 4 steps, less steps, simple operation and easy operation even for beginners.
2. The separating liquid has mild formula, does not contain detergent, does not damage mitochondrial membranes, and is favorable for maintaining the energy metabolism activity of mitochondria.
3. The separation liquid can be stored for 1 month at 4 ℃ and 6 months at 20 ℃ below zero, can be used for multiple times after being prepared once, and is convenient to use.
5. According to the invention, the Teflon grinder is connected with the IKA electric stirrer to replace a DOUNCE grinder to grind manually, so that the operation time can be shortened, the time and the labor are saved, and the requirement of simultaneously processing a large number of samples can be met. The grinding frequency can be adjusted in time by observing the cell breakage rate in the grinding step, so that the final mitochondrial yield is improved.
6. The invention has low highest centrifugal speed, low requirement on centrifugal equipment and few centrifugation times, and can simply and efficiently obtain complete mitochondria with energy metabolism activity from cultured cells.
7. The separation method of the invention has short time consumption for separating mitochondria and is beneficial to protecting the activity of mitochondria.
8. The cultured cells are animal cells, preferably mammalian cells, more preferably rat hepatic stellate cells, the cell volume is small (the grinding times are increased to 200 times by adopting DOUNCE grinding to obtain the cell breakage rate of more than or equal to 50 percent), and a large amount of complete mitochondria with energy metabolism activity can be obtained by adopting the separation method.
8. The invention has low cost, and the used reagents are common reagents and have low price.
Drawings
FIG. 1 is a diagram of a grinding process;
FIG. 2 is a graph showing cell breakage rate observed by trypan blue staining when Teflon was ground 5 times;
FIG. 3 is a graph showing the cell breakage rate observed by 10 times of grinding with Teflon and staining with Trypan blue;
FIG. 4 is a 160-fold magnification of rat hepatic stellate cells (HSC-T6);
FIG. 5 is a 160-fold enlarged view of human renal cortex proximal tubular epithelial cells (HK-2);
FIG. 6 is a 160-fold enlarged view of human glomerular endothelial cells (HRGEC);
FIG. 7 is a graph showing the cell disruption rate observed by 40 times of grinding DOUNCE with trypan blue staining;
FIG. 8 is a graph showing the cell breakage rate observed by grinding DOUNCE 80 times with trypan blue staining;
FIG. 9 is a graph showing the cell breakage rate observed by 120 times of grinding DOUNCE with Trypan blue staining,
FIG. 10 is a graph showing the cell disruption rate observed by 200 times of grinding DOUNCE with trypan blue staining;
FIG. 11 is a graph showing the ATP levels in effect example 1;
FIG. 12 is a diagram showing the mitochondrial purity at different centrifugation speeds in effect example 2.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
A method of isolating mitochondria comprising:
1) separating and resuspending the cultured cells by using a separating medium to obtain a cell suspension; the separating liquid is used for re-suspending and culturing the cells to maintain the energy metabolism activity of the cells;
2) a Teflon grinder connected to the IKA stirrer grinds the cell suspension to break the cell membrane without damaging intracellular organelles;
3) centrifuging to obtain a supernatant;
4) centrifuging the supernatant, and collecting precipitate.
Preferably, the steps 1) to 4) are performed on ice.
Preferably, the method further comprises: the centrifuge was precooled to 4 ℃ before centrifugation.
Preferably, the cultured cells are animal cells.
Preferably, the cultured cells are mammalian cells.
Preferably, the cultured cells are hepatic stellate cells.
Preferably, the cultured cells are rat hepatic stellate cells.
Preferably, the step 1) specifically comprises: for an adherent cell culture system, the culture medium is sucked from the culture system, and the separation liquid is adopted to rinse the cultured cells; collecting cells by adopting a scraping method and re-suspending the cells by adopting the separation liquid to culture the cells to obtain a cell suspension; or
For suspension cell culture systems, cultured cells are directly collected by centrifugation, rinsed with the separation solution, and resuspended with the separation solution.
Preferably, the step 1) specifically comprises:
A) for an adherent cell culture system, placing the culture system containing culture cells and a culture medium in a sample container, sucking and discarding the culture medium by a liquid transfer gun, rinsing by adopting the separating solution, and discarding the separating solution;
B) adding the separation liquid, scraping off the target cells on the wall of the sample container by using a cell scraper, and collecting the cells;
C) adding the separation solution, and collecting the residual cells;
D) combining the cells collected in step B) and the remaining cells collected in step C) to obtain a cell suspension.
Preferably, the addition amount of the separation liquid in the step B) and the step C) is 2mL/75cm2。
Preferably, the sample container is a petri dish or a culture flask.
Preferably, the sample container is a T75 culture flask.
Preferably, the separation liquid contains saccharides, EGTA, HEPES, and serum albumin.
Preferably, the final concentration of the saccharides in the separation liquid is 278-320 mM, the final concentration of EGTA is 0.1-1 mM, the final concentration of HEPES is 5-10 mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the sugar is sucrose and/or mannitol, and the animal serum albumin is bovine serum albumin.
Preferably, the final concentration of sucrose in the separation solution is 320mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the final concentration of sucrose in the separation solution is 70mM, the final concentration of mannitol is 210mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
Preferably, the bovine serum albumin is fatty acid-free bovine serum albumin.
Preferably, the separation medium contains sucrose at a final concentration of 320mM, EGTA at a final concentration of 1mM, HEPES at a final concentration of 10mM, and fatty acid-free bovine serum albumin at a final concentration of 0.5% (g/mL).
Preferably, the pH value of the separation liquid is 7.2.
Preferably, the preparation method of the separation liquid specifically comprises the following steps:
mixing sugar, EGTA, HEPES, animal serum albumin and water;
and adjusting the pH value.
Preferably, the preparation method of the separation liquid specifically comprises the following steps:
mixing sucrose, EGTA, HEPES, fatty acid-free bovine serum albumin and water; the final concentration of sucrose in the separating medium is 320mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5% (g/mL);
the pH was adjusted to 7.2 using KOH.
Preferably, the separation solution is formulated to be stored at 4 ℃ for 1 month and at-20 ℃ for 6 months.
Preferably, the stirring speed of the stirring treatment in the step 2) is 300-500 rpm.
Preferably, the stirring speed of the stirring treatment in the step 2) is 350 revolutions per minute.
Preferably, the number of times of up-and-down grinding is more than or equal to 3, and the cell breakage rate of the cell suspension is more than or equal to 50%.
Preferably, the number of times of up-and-down grinding is 10.
Preferably, cell disruption rate is observed using trypan blue staining.
Preferably, in the step 3), the rotating speed is 700-1000 Xg, and the centrifugation is carried out for 5-10 min at the temperature of 0-4 ℃;
and 4) centrifuging at the rotating speed of 3000-6000 Xg in the step 4) for 15min at the temperature of 0-4 ℃.
Preferably, the centrifugation treatment in the step 3) is performed at the rotation speed of 700Xg for 10min at 4 ℃.
Preferably, the centrifugation treatment in the step 4) is performed at the rotating speed of 3000Xg for 15min at the temperature of 4 ℃.
Preferably, the method further comprises: adding separated liquid to resuspend mitochondria for activity detection.
Interpretation of terms: energy metabolism activity of mitochondria refers to the ability of mitochondria to undergo oxidative phosphorylation. Mitochondria are energy factories of the body, and 5 complexes on the inner membrane constitute the electron transport chain, which is the material and structural basis for energy metabolism, i.e., oxidative phosphorylation, of mitochondria. The separated mitochondria not only need to keep the structural integrity and not be damaged, but also need to ensure that the complex activity of an electron transfer chain is not lost, can be oxidized and phosphorylated and have energy metabolism activity.
In the embodiment of the invention, sucrose, EGTA, HEPES and fatty acid-free bovine serum albumin are all commercial products.
Example 1
Method for separating mitochondria
1. Preparing a separation liquid:
mixing sucrose, EGTA, HEPES, fatty acid-free bovine serum albumin and water; the final concentration of sucrose in the separating medium is 320mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5% (g/mL);
the pH was adjusted to 7.2 using KOH.
The prepared separation liquid can be stored at 4 ℃ for 1 month and at-20 ℃ for 6 months.
2. Preparing ice, pre-cooling the centrifuge to 4 ℃, and finishing the following steps (step 3) on the ice, and keeping the temperature at 0-4 ℃.
3. Collecting cells
A) Taking an adherent cell culture system, placing the culture system containing culture cells and a culture medium in a T75 culture bottle, sucking and discarding the culture medium by using a liquid transfer gun, rinsing by using a separating solution, and discarding the separating solution;
B) 2mL/75cm was added2Separating liquid, scraping off target cells on the wall of a sample T75 culture flask by using a cell scraper, and collecting the cells;
C) 2mL/75cm was added2Separating the solution, and collecting the residual cells;
D) combining the cells collected in step B) with the remaining cells collected in step C) to obtain a cell suspension;
wherein the cultured cells are rat hepatic stellate cells (HSC-T6), and the culture medium is a hepatic stellate cell culture medium.
4. Grinding:
as shown in FIG. 1 (grinding process diagram, the steps are completed on ice, the connection between the grinder and the stirrer is clearly shown when photographing, and no ice bath is placed), the cell suspension is transferred into a glass tube of a Teflon grinder, the Teflon grinder is connected with an IKA stirrer, the cell suspension is stirred at the rotating speed of 350 r/min, and the grinding times are 10 times up and down, so as to obtain a mixture;
after grinding, observing the cell breakage rate through trypan blue staining to observe the cell breakage rate, and when the breakage rate is less than 50%, increasing the grinding times (the total up-and-down grinding times are 10 times (the grinding times are changed according to different cells, the cell volume of the rat hepatic stellate cell used in the embodiment is smaller, so the grinding times are mostly 10 times) until the cell breakage rate of the cell suspension is more than or equal to 50%, thus obtaining the cell suspension with the cell breakage rate more than or equal to 50%.
The cell breakage rate observed by trypan blue staining when Teflon was ground 5 times is shown in FIG. 2, and the cell breakage rate observed by trypan blue staining when Teflon was ground 10 times is shown in FIG. 3. In FIGS. 2 and 3, the broken cells are blue dots, and the unbroken cells are bright dots.
The grinding times vary according to different cells, the cells used in this example are rat hepatic stellate cells, the volume of the cells is small, so that the grinding times are 10 times to obtain a cell suspension with a cell breakage rate of not less than 50%, and other cells with larger volume only need to be ground for 3-5 times.
5. Centrifuging:
transferring the mixture into a 4mL centrifuge tube, centrifuging at the rotation speed of 700Xg at 4 ℃ for 10min to precipitate large organelles such as unbroken cells, cell fragments and cell nucleuses, and collecting supernatant to a new centrifuge tube;
6. centrifuging:
centrifuging the supernatant at 4 deg.C for 15min at 3000Xg, collecting precipitate as mitochondria, and discarding the supernatant.
The total time of the steps 2 to 6 is 30 to 50 min.
As can be seen from FIGS. 2 and 3, most of the hepatic stellate cells of rats with small cell volumes can be crushed by grinding the hepatic stellate cells up and down for 10 times, so that the method is time-saving and labor-saving, and can meet the requirement of simultaneously processing a large number of samples.
In addition, comparing the sizes of different cells, FIG. 4 is a 160-fold enlarged graph of rat hepatic stellate cells (HSC-T6) and the cell volume is smaller, FIG. 5 is a 160-fold enlarged graph of human renal cortex proximal tubular epithelial cells (HK-2), and FIG. 6 is a 160-fold enlarged graph of human glomerular endothelial cells (HRGEC). It can be seen that the size of the hepatic stellate cells of the rat adopted by the invention is smaller.
Example 2
This example differs from example 1 only in step 1, preparation of the separation: the final concentration of sucrose in the separation solution is 278mM, the final concentration of EGTA is 0.1mM, the final concentration of HEPES is 5mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5% (g/mL)
Example 3
This example differs from example 1 only in step 1, preparation of the separation:
mixing sucrose, mannitol, EGTA, HEPES, fatty acid-free bovine serum albumin and water; the final concentration of sucrose in the separating medium is 70mM, the final concentration of mannitol is 210mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5% (g/mL).
Example 4
This example differs from example 1 only in step 4, grinding: stirring at 300 revolutions per minute.
Example 5
This example differs from example 1 only in step 4, grinding: stirring at 500 rpm.
Example 6
This example differs from example 1 only in step 5, centrifugation: rotating at 1000Xg, centrifuging at 0 deg.C for 5min to precipitate large organelles such as unbroken cell, cell fragment and cell nucleus, and collecting supernatant to new centrifuge tube.
Example 7
This example differs from example 1 only in step 6, centrifugation: centrifuging the supernatant at 0 deg.C for 15min at 3000Xg, collecting precipitate as mitochondria, and discarding the supernatant.
Example 8
The difference between the present embodiment and embodiment 1 is only that the centrifugal rotation speed is different in step 6, and step 6 is:
centrifuging the supernatant at 6000Xg at 4 deg.C for 15min, collecting precipitate as mitochondria, and discarding the supernatant.
Example 9
The difference between the present embodiment and embodiment 1 is only that the centrifugal rotation speed is different in step 6, and step 6 is:
centrifuging the supernatant at 9000Xg at 4 deg.C for 15min, collecting precipitate as mitochondria, and discarding the supernatant.
Comparative example 1
Technical problem the technical solution adopted is to obtain mitochondria using a mitochondrial isolation kit (ThermoFisher-89874)
Counting the number of cells by adopting a cell mitochondria extraction kit (ThermoFisher-89874) according to an operation manual of the kit, and determining the dosage of a separation reagent; preparing a separation reagent on ice: the separating agent A is a lysis solution and the separating agent C is a termination reaction solution
The following operations were all carried out on ice:
1. taking 1mL of separation reagent A/sample and 1.3mL of separation reagent C/sample, and adding a protease inhibitor (1mL of A solution +10 mu l of PMSF)) into the A solution before use;
2. placing a culture system containing cultured cells and a culture medium into a T75 culture bottle, sucking away the culture medium, rinsing with precooled PBS, removing PBS, adding trypsin for digestion until the cells become round and fall off, adding the culture medium for termination, collecting the cells to obtain a mixture, centrifuging at 850Xg and 4 ℃ for 2 minutes, sucking away supernatant to obtain cell sediment, adding precooled PBS for resuspension of the cells, centrifuging, sucking away the supernatant to obtain cell sediment; directly collecting the mixture of the cells and the culture medium if the cells are suspended cells, centrifuging the mixture at 850Xg and 4 ℃ for 2 minutes to obtain cell precipitates, sucking and discarding supernatant, adding precooled PBS (phosphate buffer solution) to resuspend the cells, centrifuging the cells, sucking and discarding the supernatant to obtain cell precipitates;
the cultured cells are rat hepatic stellate cells (HSC-T6), and the culture medium is hepatic stellate cell culture medium
3. Discarding the supernatant, adding 800 μ L of separation reagent A/sample, oscillating at medium speed for 5 seconds, and standing on ice for 2 minutes to obtain cell suspension;
4. transferring the cell suspension into a DOUNCE grinder, grinding the cell suspension up and down, transferring the cell suspension into a centrifuge tube, adding 800 mu L of separation reagent C/sample, adding the remaining 200 mu L of separation reagent A into the DOUNCE grinder, washing, collecting and combining the separation reagent A and the sample into the centrifuge tube;
the DOUNCE grinder grinds 40-80 times recommended by the instruction, and the grinding times are set to be 40 times, 80 times, 120 times and 200 times in the comparison example;
5. centrifuging: 700Xg, centrifugation at 4 ℃ for 10 minutes;
6. taking the supernatant, transferring the supernatant into a new centrifugal tube, and precipitating the supernatant into unbroken cells, cell fragments, cell nucleuses and the like;
7. centrifuging: 3000-12000Xg, centrifugation at 4 ℃ for 15 minutes;
8. the supernatant was discarded and the mitochondria precipitated, 500. mu.L of isolation reagent C/sample was added, 12000Xg, and centrifuged at 4 ℃ for 5 minutes.
The cell breakage rate observation graph of 40 times trypan blue staining of DOUNCE grinding is shown in FIG. 7, the cell breakage rate observation graph of 80 times trypan blue staining of DOUNCE grinding is shown in FIG. 8, the cell breakage rate observation graph of 12 times trypan blue staining of DOUNCE grinding is shown in FIG. 9, and the cell breakage rate observation graph of 200 times trypan blue staining of DOUNCE grinding is shown in FIG. 10. In FIGS. 7-10, the broken cells are blue dots and the unbroken cells are light dots.
As can be seen from FIGS. 7-10, a large number of unbroken cells exist even for 200 times of hepatic stellate cells of rats with small cell volumes, which is time-consuming and labor-consuming and cannot meet the requirement of simultaneously processing a large number of samples.
Effect example 1
1. The mitochondrial fraction 2 obtained was isolated using the method of example 1 and tested:
group 1 mitochondria were those of untreated resting HSC-T6;
group 2 mitochondria were activated using TGF- β, activated HSC-T6 mitochondria were activated using TGF- β.
2 equal amounts of mitochondria were assayed for ATP levels using luminescense atpdetection assaykit (Abcam, ab113849) and 2 sets of data were counted using graphpadprism 7.02.
2. The mitochondrial fraction 2 separated by the method of comparative example 1 was tested according to the procedure of effect example 1, and the mitochondria of fraction 1 were untreated mitochondria of HSC-T6 in a resting state;
group 2 mitochondria were activated using TGF- β, activated HSC-T6 mitochondria were activated using TGF- β.
2 equal amounts of mitochondria were assayed for ATP levels using luminescense atpdetection assaykit (Abcam, ab113849) and 2 sets of data were counted using graphpadprism 7.02.
4. The results are shown in FIG. 11.
In FIG. 11 the data for mitochondria of example 1 are presented. Wherein qHSC-T6 is an untreated resting HSC-T6 result and the ATP level is 127.4 +/-5.657 (n is 3); in FIG. 11, aHSC-T6 is the result of activated HSC-T6 induced activation using TGF-. beta.and ATP levels were 269.7. + -. 15.51(n ═ 3).
FIG. 11 shows mitochondrial data for control 1, where qHSC-T6 is an untreated resting HSC-T6 result with ATP levels of 73.39 + -8.803 (n-3); in FIG. 11, aHSC-T6 is the result of activated HSC-T6 induced by TGF-. beta.activation, and ATP levels were 135.4. + -. 7.277 (n. sup.3).
The result shows that the ATP level is increased after HSC-T6 is activated, which indicates that the invention can rapidly separate out complete mitochondria from cells and maintain the energy metabolism activity, and the ATP level of the invention is obviously higher than that of the control example 1.
Effect example 2
The mitochondrial purity obtained in examples 1, 8 and 9 was examined and the results are shown in fig. 12 (graph of mitochondrial purity at different speeds), where PMP70 is a peroxisome marker, which is close to mitochondria and therefore susceptible to contamination, PCNA is a nuclear marker, GAPDH is a cytoplasmic marker, and GRP75 is a mitochondrial marker, which reflects consistent loading of mitochondrial proteins.
As can be seen from the figure, the rotating speed of the cleaner reaches 6000, so that the cleaning speed is 3000-6000 Xg, preferably 3000 Xg.
Namely, the highest centrifugal speed is low, the highest centrifugal speed can be 3000Xg, the requirement on centrifugal equipment is low, the centrifugal frequency is low, and complete mitochondria with energy metabolism activity can be simply and efficiently obtained from cultured cells.
The prior art mainly comprises two methods for separating and culturing mitochondria in cells, one method is to use a DOUNCE glass grinder to break the cells, and the other method is based on the principle of gradient separation, wherein the former method is a scheme adopted by a commercial kit.
Firstly, use DOUNCE glass grinder to break cells
The commercial kit in the comparative example 1 is expensive, and the price is 3000-7000 yuan/50 samples; the components are all liquid, and the shelf life is 1 year; ③ 3 components in the kit of the comparative example 1 all contain detergent to promote cell rupture, and the existence of trace detergent can destroy the integrity of mitochondrial membranes; the DOUNCE grinder is a manual grinder, and the cells are ground for dozens of times when being sufficiently broken, thereby wasting time and labor.
The reagent used in the embodiment 1 is low in price; ② the powder form, the shelf life is as long as 3 years; the reagent does not contain any detergent, has mild components, and does not damage mitochondrial membranes, thereby being beneficial to maintaining the energy metabolism activity of mitochondria; the Teflon grinder can be directly connected with the IKA cantilever stirrer, electric grinding replaces manual grinding, most of cells can be broken by grinding hepatic stellate cells of rats with small cell volumes up and down for 10 times, time and labor are saved, and the requirement for simultaneously processing a large number of samples can be met.
Second, based on gradient separation principle to separate mitochondria
The technology based on the gradient separation principle needs an ultracentrifuge capable of ultracentrifuging, and has high requirements on equipment; carefully operating when the prepared gradient sucrose solution is added, otherwise, mixing different gradient sucrose; thirdly, when the gradient layer containing mitochondria is finally sucked, the mitochondria loss can be caused because the sucking is not complete, and the yield is reduced; if Percoll gradient centrifugation is used, the cost is high; long operation time, basically 1 hour.
The centrifugal rotating speed is only 3000xg at most, and the requirement of a common low-temperature centrifugal machine can be met; secondly, the operation is simple, and only separation liquid is needed in the whole process; thirdly, the mitochondria are finally a precipitate part, and the supernatant is sucked and discarded without loss; reagents used are all conventional reagents, and the price is low; the whole process only needs 30-50min, which is more beneficial to protecting the activity of mitochondria.
In addition, the energy metabolism activity of mitochondria is reduced along with the time extension, the activity difference of different groups can not be detected after more than 3-4 hours, the operation time of the prior art is generally more than or equal to 1 hour, the centrifugation times are more than that of the invention, and the operation time of other methods is prolonged more than that of the invention along with the increase of the processed sample size.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
1. A method for separating mitochondria, comprising:
1) re-suspending the cultured cells by using the separation liquid to obtain a cell suspension;
2) the Teflon grinder is connected to the IKA stirrer to grind the cell suspension;
3) centrifuging to obtain a supernatant;
4) centrifuging the supernatant, and collecting precipitate.
2. The method of claim 1, wherein steps 1) through 4) are performed on ice.
3. The method of claim 1, wherein the cell is a rat hepatic stellate cell.
4. The method according to claim 1, wherein the step 1) specifically comprises: for an adherent cell culture system, the culture medium is sucked from the culture system, and the separation liquid is adopted to rinse the cultured cells; collecting cells by adopting a scraping method and re-suspending the cells by adopting the separation liquid to culture the cells to obtain a cell suspension; or
For suspension cell culture systems, cultured cells are directly collected by centrifugation, rinsed with the separation solution, and resuspended with the separation solution.
5. The method according to claim 4, wherein the step 1) specifically comprises:
A) for an adherent cell culture system, placing the culture system containing culture cells and a culture medium in a sample container, sucking and discarding the culture medium by a liquid transfer gun, rinsing by adopting the separating solution, and discarding the separating solution;
B) adding the separation liquid, scraping off the target cells on the wall of the sample container by using a cell scraper, and collecting the cells;
C) adding the separation solution, and collecting the residual cells;
D) combining the cells collected in step B) and the remaining cells collected in step C) to obtain a cell suspension.
6. The method according to claim 1, wherein the separation liquid contains saccharides, EGTA, HEPES, and serum albumin.
7. The method according to claim 6, wherein the final concentration of the saccharide in the separation solution is 278-320 mM, the final concentration of EGTA is 0.1-1 mM, the final concentration of HEPES is 5-10 mM, and the final concentration of animal serum albumin is 0.5% (g/mL).
8. The method according to claim 1, wherein the stirring speed in the stirring treatment in the step 2) is 300 to 500 rpm.
9. The method of claim 1, wherein the number of times of grinding is not less than 3.
10. The method according to claim 1, wherein the centrifugation treatment in step 3) is performed at a rotation speed of 700-1000 Xg and at 0-4 ℃ for 5-10 min;
and 4) centrifuging at the rotating speed of 3000-6000 Xg in the step 4) for 15min at the temperature of 0-4 ℃.
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