CN113186161A - Method for quickly separating ultra-high purity human blood neutrophils - Google Patents
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Abstract
The invention relates to a method for rapidly separating human blood neutrophils with ultrahigh purity, which comprises the following steps: firstly, adding the neutrophil separating medium into a separating tube, and then adding the human whole blood above the neutrophil separating medium to form a whole blood layer; centrifuging the separation tube to divide the liquid in the tube into 5 layers, and collecting a neutrophilic granulocyte layer and liquid transiting from the neutrophilic granulocyte layer to a red blood cell layer; diluting, centrifuging and discarding the supernatant; lysing erythrocytes and centrifuging and discarding the supernatant; and (4) sorting the neutrophils by magnetic beads. The invention can greatly solve the influence of other immune cells on the neutrophil, so that the purity of the neutrophil separation is stable and is more than 99%, and the separation time is reduced (about 1 hour in the whole process), thereby providing a good technical platform for the intervention of the neutrophil inflammatory reaction of clinical infectious diseases.
Description
Technical Field
The invention relates to a method for quickly separating human blood neutrophils with ultrahigh purity, belonging to the technical field of human blood neutrophils separation.
Background
To the best of the applicant's knowledge, neutrophils are the most abundant immune cell type in the host blood and play a first line of defense in defending against the invasion of many pathogens. The neutrophil granulocytes have active deformation movement and phagocytosis functions and play a very important role in defense; the phagocytic object mainly takes bacteria as main object and also phagocytizes foreign matters. Neutrophils are produced in the bone marrow, pass through promyelocytes, mesogranules, promyelocytes, rod-shaped nuclei and lobular nuclei and are ultimately released in mature form into peripheral blood or tissues. It should be noted that when the pathogen load exceeds the load of immune cells, an inflammatory cascade reaction is triggered, which poses a great threat to life, while in many clinical anti-infective treatments, most of them only intervene on pathogenic microorganisms, and the intervention on immune cell activation and the inflammatory factor cascade effect caused by immune cells is less and less effective. The immune cells are various in types and interact with each other, and as the most abundant cell types of the immune cells, the intervention on the maximum subgroup of the immune cells is particularly important. Therefore, obtaining neutrophil with ultra-high purity (purity more than 99%) is an urgent need for the targeted research of immune cell inflammatory cascade reaction, and is also the basis of the theory of neutrophil inflammatory reaction.
The current methods for neutrophil isolation are: gradient centrifugation (e.g., Percoll discontinuous density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation), erythrocyte lysis, and Dextran cell sedimentation, but have many disadvantages. The gradient separation method is used more at present, but the method has the defects that the method is mixed with mononuclear cells and the like during cell gradient layering, the quantity of neutrophils obtained in the later period is small, and more mixed cells exist; although the erythrocyte lysis method is simple, a large amount of immune cells are mixed, the purity is low, and the erythrocyte lysis method is not suitable for targeted intervention of immune cell inflammation; the Dextran cell sedimentation method has the condition that the separation time is long, and after separation, neutrophils are activated and even die. Therefore, the development of a technical means for rapidly separating and obtaining human blood neutrophils with ultra-high purity is urgently needed.
It has been found through the search that patent No. CN201280057552.5, patent publication No. CN103946373B, discloses a method for separating cells from a sample containing red blood cells, which method recovers desired cells from a sample containing desired cells, red blood cells and undesired cells, comprising:
a) contacting the sample with a composition comprising: i) a red blood cell agglutination reagent, ii) at least one antigen recognition moiety coupled to the magnetic particle, wherein the particle having the at least one antigen recognition moiety specifically binds to at least one antigen specific for one or more unwanted cellular components; b) simultaneously applying i) gravity sedimentation for erythrocyte sedimentation and ii) a magnetic field gradient to the sample for immobilizing the magnetic particles, thereby producing a pellet and a supernatant phase, and c) recovering the desired cells from the supernatant phase. However, this solution is not specifically designed for neutrophils and is costly.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the method for rapidly separating the human blood neutrophils with ultrahigh purity is provided, the human blood neutrophils with ultrahigh purity can be rapidly separated and obtained, and the purity of the human blood neutrophils is more than 99%, so that a technical platform is provided for the research of neutrophilic granulocythemia inflammation.
The technical scheme for solving the technical problems of the invention is as follows:
a method for rapidly separating ultra-high purity human blood neutrophils is characterized by comprising the following steps:
firstly, adding a neutrophilic granulocyte separating medium into a separating tube, and adding human whole blood above the neutrophilic granulocyte separating medium to form a whole blood layer;
secondly, centrifuging the separation tube to divide the liquid in the tube into 5 layers, wherein each layer comprises a plasma layer, a cell separation liquid layer, a monocyte layer, a neutrophil layer and a red blood cell layer from top to bottom in sequence, wherein the plasma layer is a top layer, and the red blood cell layer is a bottom layer; discarding a plasma layer, a cell separation liquid layer and a monocyte layer, collecting a neutrophilic granulocyte layer and liquid transiting from the neutrophilic granulocyte layer to the erythrocyte layer into a clean separation tube, and discarding the erythrocyte layer as a bottom layer;
thirdly, diluting and centrifuging the liquid obtained in the second step, and then removing supernatant to obtain cell masses; adding erythrocyte lysate and suspending cells, and placing on ice for lysis; then centrifuging to remove supernatant and resuspending cells;
fourthly, adopting a neutrophil magnetic bead sorting kit, taking antibodies in the kit, adding the antibodies into the resuspended cells obtained in the third step, and reversing and uniformly mixing the antibodies; adding magnetic beads, reversing and uniformly mixing; inserting the separation tube into a magnet frame and standing; after standing, transferring the buffer solution containing the cells in the separating tube to a clean separating tube to obtain the human blood neutrophils with ultrahigh purity.
The method can greatly solve the influence of other immune cells on the neutrophil, so that the purity of the neutrophil separation is stable and is more than 99%, and the separation time is reduced (about 1 hour in the whole process), thereby providing a good technical platform for the intervention of the neutrophil inflammatory reaction in clinical infectious diseases and having important application value.
The technical scheme of the invention is further perfected as follows:
preferably, before the first step, all reagents are left standing at room temperature, and after the temperature of each reagent is stabilized, separation is performed from the first step; the room temperature refers to the ambient temperature.
With this preferred embodiment, the subsequent separation process can be carried out more smoothly.
Preferably, in the first step, the volume ratio of the neutrophil separating medium to the human whole blood is 1 ± 0.1: 1; in the second step, the centrifugal condition is 500 plus or minus 10g/min for 20-30min, and the centrifugal brake is set to be 0.
More preferably, in the third step, the dilution is performed by using HBSS buffer solution without calcium and magnesium, and the volume ratio of the HBSS buffer solution to human whole blood is 2 +/-0.1: 1, reversing and mixing evenly for several times to suspend the cells; centrifuging at 350 + -5 g/min for 10-15 min; the volume ratio of the erythrocyte lysate to the human whole blood is 1.6 +/-0.1: 1; the cracking time is 5-8min when the glass is placed on ice; centrifuging at 250 + -5 g/min for 5-10 min; and (3) adopting HBSS buffer solution without calcium and magnesium when the cells are re-suspended, wherein the volume ratio of the HBSS buffer solution to human whole blood is 0.6 +/-0.1: 1, after resuspending the cells, the whole liquid was transferred to a clean separation tube.
More preferably, the fourth stepThe ratio of antibody volume to cell concentration was 5 ± 0.5 μ Ι: 5 +/-0.5X 106Per ml; the total volume of the added beads was the same as the volume of the antibody.
More preferably, in the fourth step, a rotary mixer is adopted when reversing and mixing uniformly, and the uniform rotation is carried out for 5-10min at the rotor speed of 12 plus or minus 2 rpm; standing for 8-10min, and opening the cover of the separation tube during standing; the separation tube is kept inserted in the magnet holder during the transfer.
More preferably, in the third step, the action of reversing the mixing is gentle and kept within the range of ensuring that the leukocytes are not activated; and in the fourth step, transferring the buffer solution containing the cells in the separation tube to a clean separation tube, adding the antibody, placing the antibody in a juxtaposition rotating mixer, rotating at a constant speed for 5-10min at the rotating speed of a rotor of 12 +/-2 rpm, adding the magnetic beads, placing the magnetic beads in the juxtaposition rotating mixer, rotating at a constant speed of 12 +/-2 rpm for 5-10min, inserting the separation tube into a magnet frame, standing for at least 5min, and transferring the separation tube to another clean separation tube to obtain the ultra-high purity human blood neutrophilic granulocytes.
More preferably, in the first step, the neutrophil isolate is HISTOPAQUE-1119, Catalog # RNBH 2808; in the third step, the erythrocyte lysate is Fisher/Gibco-Catalog # A1049201; in the fourth step, the neutrophil magnetic bead sorting kit is Stem cell kit-Catalog #17957, the antibody is a Cocktail antibody, and the magnet rack is easy SepTM Magnet-Catalog#18000。
By adopting the preferred scheme, the specific parameters and the specific details of each step can be further optimized. Wherein in the third step the neutrophil activity is susceptible if the erythrocyte lysis time is longer than a defined time; in the fourth step, the mixing is reversed to bring the antibody into sufficient contact with the cells so as not to reduce the purity of the resulting neutrophils.
Preferably, in the first step, the separation tube is kept free from shaking during the process of adding the whole human blood; in the second step, if the layering is not clear after centrifugation, the centrifugation step is repeated until the layering is clear; in the third step, the lysis step is repeated once if insufficient lysis is found after resuspending the cells.
With this preferred embodiment, the separation effect can be further ensured.
Preferably, the method further comprises a cell purity identification step after the fourth step.
Note: the above time units min are minutes. Setting the centrifugal brake to 0 means that there is no brake to naturally stall at the end of centrifugation.
Compared with the prior art, the method can greatly solve the influence of other immune cells on the neutrophil, so that the purity of the neutrophil separation is stable and is more than 99%, and the separation time (about 1 hour in the whole process) is reduced, thereby providing a good technical platform for the intervention of the neutrophil inflammatory reaction of clinical infectious diseases and having important application value.
Drawings
FIG. 1 is a graph showing the flow cytometry results of example 2 of the present invention.
Detailed Description
The invention is described in further detail below with reference to embodiments and with reference to the drawings. The invention is not limited to the examples given.
Example 1
This example is a rapid separation of human blood neutrophils of ultra-high purity.
The basic process of the embodiment includes:
before the first step, all reagents are stood at room temperature, and after the temperature of each reagent is stable, separation is carried out from the first step; room temperature refers to ambient temperature.
Firstly, adding a neutrophilic granulocyte separating medium into a separating tube, and adding human whole blood above the neutrophilic granulocyte separating medium to form a whole blood layer;
wherein the volume ratio of the neutrophil separating medium to the human whole blood is 1 +/-0.1: 1; the separating tube is kept not to shake during the process of adding the whole blood of the human body.
Secondly, centrifuging the separation tube to divide the liquid in the tube into 5 layers, wherein each layer comprises a plasma layer, a cell separation liquid layer, a monocyte layer, a neutrophil layer and a red blood cell layer from top to bottom in sequence, wherein the plasma layer is a top layer, and the red blood cell layer is a bottom layer; discarding a plasma layer, a cell separation liquid layer and a monocyte layer, collecting a neutrophilic granulocyte layer and liquid transiting from the neutrophilic granulocyte layer to the erythrocyte layer into a clean separation tube, and discarding the erythrocyte layer as a bottom layer;
wherein the centrifugation condition is 500 +/-10 g/min for 20-30min, and the centrifugal brake is set to be 0; and if the layering is unclear after centrifugation, repeating the centrifugation step until the layering is clear.
Thirdly, diluting and centrifuging the liquid obtained in the second step, and then removing supernatant to obtain cell masses; adding erythrocyte lysate and suspending cells, and placing on ice for lysis; then centrifuging to remove supernatant and resuspending cells;
wherein, HBSS buffer solution without calcium and magnesium is adopted for dilution, and the volume ratio of the HBSS buffer solution to human whole blood is 2 +/-0.1: 1, reversing and uniformly mixing for a plurality of times to suspend the cells, wherein the action range of the reversing and uniformly mixing needs to be gentle and is kept within the range of ensuring that the white blood cells are not activated; centrifuging at 350 + -5 g/min for 10-15 min; the volume ratio of the erythrocyte lysate to the human whole blood is 1.6 +/-0.1: 1; the cracking time is 5-8min when the glass is placed on ice; centrifuging at 250 + -5 g/min for 5-10 min; and (3) adopting HBSS buffer solution without calcium and magnesium when the cells are re-suspended, wherein the volume ratio of the HBSS buffer solution to human whole blood is 0.6 +/-0.1: 1, after resuspending the cells, the whole liquid was transferred to a clean separation tube. If insufficient lysis is found after resuspending the cells, the lysis step is repeated once.
Fourthly, adopting a neutrophil magnetic bead sorting kit, taking antibodies in the kit, adding the antibodies into the resuspended cells obtained in the third step, and reversing and uniformly mixing the antibodies; adding magnetic beads, reversing and uniformly mixing; inserting the separation tube into a magnet frame and standing; after standing, transferring the buffer solution containing the cells in the separation tube to a clean separation tube to obtain the human blood neutrophils with ultrahigh purity;
wherein the ratio of antibody volume to cell concentration is 5 ± 0.5 μ l: 5 +/-0.5X 106Per ml; the total volume of the added magnetic beads is the same as the volume of the antibody; when the mixture is reversed and mixed, a rotary mixer is adopted, and the mixture is rotated at a constant speed for 5-10min at the rotor speed of 12 +/-2 rpm; standing for 8-10minThe separating tube is kept uncovered during the process; keeping the separating tube inserted in the magnet frame in the transferring process; transferring the buffer solution containing the cells in the separation tube to a clean separation tube, adding the antibody, placing the antibody in a rotary mixer, rotating at a constant speed for 5-10min at the rotating speed of a rotor of 12 +/-2 rpm, adding the magnetic beads, placing the magnetic beads in the rotary mixer, rotating at a constant speed of 12 +/-2 rpm for 5-10min, inserting the separation tube into a magnet frame, standing for at least 5min, and transferring the separation tube to another clean separation tube to obtain the human blood neutrophilic granulocytes with ultrahigh purity.
The following is a specific procedure by way of example:
(1) the specific raw material substances adopted.
Human whole blood 5ml, neutrophil lysate (HISTOPAQUE-1119, Catalog # RNBH2808), 15ml transparent centrifuge Tube (Falcon Tube), HBSS buffer solution without calcium and magnesium, erythrocyte lysate (Fisher/Gibco-Catalog # A1049201), 5ml separation Tube (Falcon Tube), magnetic bead sorting kit (Stem cell kit-Catalog #17957), magnet rack (EasySep)TM Magnet-Catalog#18000)。
(2) The method comprises the following specific steps:
1. all reagents were placed at room temperature and after temperature stabilization, neutrophil isolation was performed.
2.5 ml of the neutrophil isolate was placed in a 15ml clear separation tube, and 5ml of whole blood was carefully added to the upper layer of the isolate, at which time the blood was clearly observed to be in the upper layer of the isolate.
3. Centrifuging at room temperature of 500g/min for 20min (note that the centrifugal brake is set to 0, the setting aims at clearer layering after centrifugation), and dividing liquid in the tube into 5 layers from top to bottom: plasma layer, cell separation layer, monocyte layer, neutrophil layer, red blood cell layer (bottom layer). Remarking: if the stratification is unclear, step 3 needs to be repeated.
4. The top three layers (plasma layer, cell separation layer, monocyte layer) were carefully removed and discarded.
5. The fluid between the neutrophil layer, the neutrophil layer and the red blood cell layer is collected and transferred to a new separation tube.
6. The cells were suspended by diluting with 10ml of HBSS buffer without calcium and magnesium and slowly inverting and mixing 3 to 5 times.
7. The cells were centrifuged at 350g/min for 10min, the cell pellet settled to the bottom of the tube (including erythrocytes and neutrophils), and the supernatant of the centrifugation was removed with a pipette.
8. Add 8ml of erythrocyte lysate, gently blow the bottom cells to suspend, and place on ice to lyse for 5 min.
9. The cells were centrifuged at 250g/min for 5min and the supernatant discarded (if lysis was insufficient, this step could be repeated once).
10. Cells were suspended by adding 3ml of HBSS buffer without calcium and magnesium. The cells were transferred to a 5ml separation tube.
11. And separating and purifying by using a magnetic bead sorting kit. Mu.l of Cocktail antibody (5. mu.l per 5X 10) was added6And/ml), evenly mixing by reversing a rotary mixer, and uniformly rotating for 5min at the rotating speed of a rotor of 12 rpm. Adding the magnetic beads with the same volume, reversing and uniformly mixing by adopting a rotary mixer, and uniformly rotating for 5min at the rotor speed of 12 rpm.
12. The tube was inserted into a magnet holder and left to stand for 8min, the process keeping the lid open.
13. The buffer containing the cells was poured into another clean separator tube (5ml) and the buffer transfer process ensured that the tubes were in the magnet rack and after transfer of the cell suspension, the original separator tube was discarded.
14. Adding the antibody into the separation tube, placing the separation tube in a rotating mixer, rotating at the rotor speed of 12rpm for 5min at a constant speed, adding the magnetic beads into the rotating mixer, rotating at the rotor speed of 12rpm for 5min at a constant speed, inserting the separation tube into a magnet frame again, standing for 5min, and transferring to another clean separation tube to obtain the human blood neutrophils.
Example 2
This example is to examine the purity of human blood neutrophils obtained in example 1.
PE anti-human CD66b Antibody (Biolegend, cat # 305105) and FITC anti-human CD16 Antibody (Biolegend, cat # 302005) were used.
The human blood neutrophil-containing liquid obtained as described in example 1 was poured into another new tube (5ml) according to the method described in example 1 and step 13, the number of cells was counted, and the cells were labeled by the double labeling method of PE anti-human CD66b Antibody and FITC anti-human CD16 Antibody, 5. mu.l per tube, incubated for 20min, and then the purity of the isolated neutrophils was confirmed by flow cytometry. As shown in FIG. 1, the purity of human blood neutrophils was stable at > 99% by flow cytometry using a double-label method of PE anti-human CD66b antibodies and FITC anti-human CD16 antibodies.
In addition, the results of identifying human blood neutrophils prepared in example 1 were the same or substantially the same as the above results, and the same conclusion as the above was drawn.
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Claims (10)
1. A method for rapidly separating ultra-high purity human blood neutrophils is characterized by comprising the following steps:
firstly, adding a neutrophilic granulocyte separating medium into a separating tube, and adding human whole blood above the neutrophilic granulocyte separating medium to form a whole blood layer;
secondly, centrifuging the separation tube to divide the liquid in the tube into 5 layers, wherein each layer comprises a plasma layer, a cell separation liquid layer, a monocyte layer, a neutrophil layer and a red blood cell layer from top to bottom in sequence, wherein the plasma layer is a top layer, and the red blood cell layer is a bottom layer; discarding a plasma layer, a cell separation liquid layer and a monocyte layer, collecting a neutrophilic granulocyte layer and liquid transiting from the neutrophilic granulocyte layer to the erythrocyte layer into a clean separation tube, and discarding the erythrocyte layer as a bottom layer;
thirdly, diluting and centrifuging the liquid obtained in the second step, and then removing supernatant to obtain cell masses; adding erythrocyte lysate and suspending cells, and placing on ice for lysis; then centrifuging to remove supernatant and resuspending cells;
fourthly, adopting a neutrophil magnetic bead sorting kit, taking antibodies in the kit, adding the antibodies into the resuspended cells obtained in the third step, and reversing and uniformly mixing the antibodies; adding magnetic beads, reversing and uniformly mixing; inserting the separation tube into a magnet frame and standing; after standing, transferring the buffer solution containing the cells in the separating tube to a clean separating tube to obtain the human blood neutrophils with ultrahigh purity.
2. The method for rapid isolation of human neutrophils with ultra-high purity according to claim 1, wherein all reagents are left standing at room temperature before the first step, and the isolation is performed from the first step after the temperature of each reagent is stabilized; the room temperature refers to the ambient temperature.
3. The method for rapidly separating human neutrophil with ultra-high purity according to claim 1, wherein in the first step, the volume ratio of the neutrophil separating solution to the whole human blood is 1 ± 0.1: 1; in the second step, the centrifugal condition is 500 plus or minus 10g/min for 20-30min, and the centrifugal brake is set to be 0.
4. The method for rapidly separating human neutrophil with ultra-high purity according to claim 3, wherein in the third step, the dilution is carried out by using HBSS buffer solution without calcium and magnesium, and the volume ratio of HBSS buffer solution to human whole blood is 2 ± 0.1: 1, reversing and mixing evenly for several times to suspend the cells; centrifuging at 350 + -5 g/min for 10-15 min; the volume ratio of the erythrocyte lysate to the human whole blood is 1.6 +/-0.1: 1; the cracking time is 5-8min when the glass is placed on ice; centrifuging at 250 + -5 g/min for 5-10 min; and (3) adopting HBSS buffer solution without calcium and magnesium when the cells are re-suspended, wherein the volume ratio of the HBSS buffer solution to human whole blood is 0.6 +/-0.1: 1, after resuspending the cells, the whole liquid was transferred to a clean separation tube.
5. The method for rapid isolation of human neutrophils having ultra-high purity according to claim 4, wherein in the fourth step, the antibody volume and cell concentrationIn a ratio of 5. + -. 0.5. mu.l: 5 +/-0.5X 106Per ml; the total volume of the added beads was the same as the volume of the antibody.
6. The method for rapidly separating human neutrophil with ultra-high purity according to claim 5, wherein in the fourth step, a rotary mixer is adopted for reverse mixing, and the mixture is uniformly rotated for 5-10min at a rotor speed of 12 plus or minus 2 rpm; standing for 8-10min, and opening the cover of the separation tube during standing; the separation tube is kept inserted in the magnet holder during the transfer.
7. The method for rapidly separating human neutrophils with ultra-high purity according to claim 6, wherein in the third step, the action of reversing and mixing is gentle and kept within the range of ensuring the inactivation of leukocytes; and in the fourth step, transferring the buffer solution containing the cells in the separation tube to a clean separation tube, adding the antibody, placing the antibody in a juxtaposition rotating mixer, rotating at a constant speed for 5-10min at the rotating speed of a rotor of 12 +/-2 rpm, adding the magnetic beads, placing the magnetic beads in the juxtaposition rotating mixer, rotating at a constant speed of 12 +/-2 rpm for 5-10min, inserting the separation tube into a magnet frame, standing for at least 5min, and transferring the separation tube to another clean separation tube to obtain the ultra-high purity human blood neutrophilic granulocytes.
8. The method for rapid isolation of human blood neutrophils of ultra-high purity according to any one of claims 2 to 7, wherein in the first step, the neutrophil isolate is HISTOPAQUE-1119, Catalog # RNBH 2808; in the third step, the erythrocyte lysate is Fisher/Gibco-Catalog # A1049201; in the fourth step, the neutrophil magnetic bead sorting kit is Stem cell kit-Catalog #17957, the antibody is a Cocktail antibody, and the magnet rack is easy SepTMMagnet-Catalog#18000。
9. The method for rapid isolation of human neutrophils with ultra-high purity according to any one of the claims 1 to 7, wherein in the first step, the isolation tube is kept free from shaking during the whole human blood addition; in the second step, if the layering is not clear after centrifugation, the centrifugation step is repeated until the layering is clear; in the third step, the lysis step is repeated once if insufficient lysis is found after resuspending the cells.
10. The method for rapid isolation of ultra-high purity human blood neutrophils according to any one of claims 1-7, wherein said method further comprises a cell purity identification step after the fourth step.
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