CN108396008B - Abalone blood cell processing method - Google Patents
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- CN108396008B CN108396008B CN201810075334.3A CN201810075334A CN108396008B CN 108396008 B CN108396008 B CN 108396008B CN 201810075334 A CN201810075334 A CN 201810075334A CN 108396008 B CN108396008 B CN 108396008B
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Abstract
The invention discloses an abalone blood cell processing method, which comprises the steps of firstly adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube; then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily; after the blood flows out, 1ml of blood is pumped by a sterile syringe and is directly injected into a centrifuge tube added with the anticoagulation protective agent; filtering the blood with a hemofilter for flow cytometry into a new 2ml centrifuge tube; centrifuging at 18 deg.C and 3000rpm for 10min, and removing supernatant; finally, 1ml of filtered seawater is used for resuspending blood cells to prepare abalone hemolymph suspension. The method adopts proper blood anticoagulation protective agent and blood filter, can effectively prolong the agglutination time of blood cells, rapidly filter impurities in blood, improve the purity of blood, reduce experiment errors and enable the experiment result to be more accurate.
Description
Technical Field
The invention relates to the technical field of abalone blood cell processing, and particularly relates to an abalone blood cell processing method.
Background
The abalone is an important mariculture variety in China due to rich nutrition and high economic value. At present, in the field of biological experiments, a unified treatment mode aiming at abalone blood cells is not provided. People often treat the blood cells of abalone in various ways. In the case of no anticoagulant protective agent, the abalone blood cells are greatly agglutinated within minutes once being separated from the body. Even abalone blood cells to which some anticoagulant protectors are added die largely in a short time. Therefore, the experiment is required to be completed in a short time, and the problems that the experiment result is greatly influenced by system errors, the experiment cost is increased, the experiment result is inaccurate and the like are caused. Therefore, it is important in the experiment to rapidly process blood cells and ensure that the abalone blood cells do not agglutinate within a certain time.
At present, shellfish (abalone) blood for flow cytometry detection does not have a complete or regular set of processing device, and people generally directly carry out flow cytometry detection on collected blood without filtering treatment. The results show that the untreated blood contains more impurities, and the presented image is a relatively chaotic and disorderly scattergram. In addition, the detector is easy to block due to the untreated cell group, and the detector needs to be cleaned for a long time (10-40min) before the sample measurement can be carried out continuously. Therefore, the detection process is delayed, and the test cost is increased (200 yuan/h) virtually. It is also necessary to use a suitable blood filter in the abalone blood cell processing process.
Disclosure of Invention
The invention aims to provide an abalone blood cell processing method, which can effectively prolong the agglutination time of blood cells, quickly filter impurities in blood, improve the purity of the blood, reduce experimental errors and enable experimental results to be more accurate by adopting a proper blood anticoagulation protective agent and a proper blood filter.
In order to achieve the purpose, the invention adopts the following technical scheme:
an abalone blood cell processing method, characterized in that the processing method comprises the following steps:
(1) firstly, adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube;
(2) then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily;
(3) after the blood flows out, 1ml of blood is pumped by a sterile syringe and is directly injected into a centrifuge tube added with the anticoagulation protective agent;
(4) filtering the liquid of step (3) with a hemofilter for flow cytometry into a new 2ml centrifuge tube;
(5) centrifuging at 18 deg.C and 3000rpm for 10min, and removing supernatant;
(6) and (4) finally, resuspending the blood cells treated in the step (5) by using 1ml of filtered seawater to prepare abalone hemolymph suspension.
In the abalone blood cell processing method, the anticoagulant protective agent comprises the following components: 2.45-2.55 g of sodium chloride, 0.9-1.1 g of EDTA, 0.22-0.24 g of 1, 4-piperazine diethyl sulfonic acid and 100ml of 0.1mM PBS.
Preferably, the anticoagulant protector consists of the following components: 2.5g of sodium chloride, 1g of EDTA, 0.23g of 1, 4-piperazine-diethylsulphonic acid and 100ml of 0.1mM PBS.
In the abalone blood cell processing method, the preparation method of the anticoagulation protective agent comprises the following steps:
(1) pouring 0.1mM PBS buffer solution into a beaker, adding solid sodium chloride powder into the beaker, stirring for 3-5 minutes until the sodium chloride is completely dissolved, and then adding 1, 4-piperazine diethylsulfonic acid powder, fully stirring and dissolving to obtain a mixed solution A;
(2) heating the mixed solution A in a 38-40 ℃ water bath, slowly adding EDTA (ethylene diamine tetraacetic acid) while stirring until the EDTA is completely dissolved to obtain a mixed solution B;
(3) the mixed solution B is stirred for 0.5 to 1 hour by a magnetic stirrer at room temperature, so that all the components are fully dissolved in PBS buffer solution;
(4) and (3) starting an ultraviolet sterilization function of the ultra-clean workbench for 30-35min, then filtering and removing impurities of the mixed solution B on the ultra-clean workbench by using a 0.45um sterile filter, putting the mixed solution B into a sterilized blue-cap bottle, and filtering and sterilizing the liquid in the blue-cap bottle by using a 0.22um sterile filter to finally obtain the abalone blood cell anticoagulant protective agent.
Preferably, in the preparation method of the abalone blood cell anticoagulant protective agent, the heating temperature of the water bath kettle in the step (2) is 40 ℃.
Preferably, in the preparation method of the abalone blood cell anticoagulant protective agent, the stirring time of the magnetic stirrer in the step (3) is 0.5 h.
The abalone blood cell anticoagulant protective agent can be stored in a sterilized blue cap bottle for later use at 4 ℃ or room temperature.
In addition, blood filter, including fixed station, montant, filter equipment and putting the thing device, put the thing device and place in filter equipment's below, filter equipment passes through the clamping ring pole and installs on the montant, and the clamping ring pole passes through the height adjusting knob and installs on the montant, and the montant is installed on the fixed station, and filter equipment includes clamping ring and lower clamping ring, goes up the clamping ring and all is connected with the clamping ring pole with lower clamping ring. And a bolting silk is arranged between the upper pressing ring and the lower pressing ring. And filtering out impurities in the blood by using bolting silk.
The upper pressing ring is provided with four pressing buckles, and the four pressing buckles are uniformly distributed on the periphery of the upper pressing ring. The four pressing buckles can firmly buckle the upper pressing ring on the lower pressing ring and can buckle the bolting silk between the upper pressing ring and the lower pressing ring.
Furthermore, a grid mesh is arranged on the upper pressing ring and provided with four square grids. The bolting silk is spatially divided into four parts by a grid mesh.
Still further, the storage device comprises a storage box and a centrifuge tube, and the centrifuge tube is placed in the storage box.
Preferably, a cylindrical groove is arranged in the storage box, and ice blocks are placed in the cylindrical groove. Ice cubes are used to maintain the low temperature environment required for blood filtration.
In the blood filter, two isolating pieces are arranged in the cylindrical groove, and the two isolating pieces are arranged in a vertical crossing manner. Through two spacers of vertical cross arrangement, will cylindric recess space go up to divide into four microgrooves, four microgrooves and four square grid one-to-one of grid net place a centrifuging tube in every microgroove to be convenient for mark sample, can not cause and obscure.
In the blood filter, the press button is made of plastic.
In the blood filter, the upper pressing ring and the lower pressing ring are made of stainless steel.
In the blood filter, the fixing table is a diamond-shaped iron fixing table. The diamond-shaped iron fixing table can ensure the stability of the invention and can not topple over due to the object placing device positioned on one side of the vertical rod.
The invention has the advantages that:
the abalone blood cells treated by the treatment method are not aggregated into clusters for a long time after being taken out due to the addition of the anticoagulation protective agent, the integrity and the independence of the cells are still kept, and impurities in the blood are greatly reduced after being filtered by a blood filter, so that the blood cells treated by the treatment method can obtain an experimental result with lower cost and more accurate data in subsequent experiments.
Drawings
FIG. 1 is a schematic view of a blood filter construction;
fig. 2 is a schematic structural view of the placement device.
Reference numerals: 1-fixed table, 2-vertical rod, 3-filtering device, 4-storage device, 5-ring pressing rod, 6-upper pressing ring, 7-lower pressing ring, 8-pressing buckle, 9-storage box, 10-centrifuge tube, 11-groove, 12-spacer, 13-grid mesh and 14-height adjusting button.
FIG. 3 shows agglutination of abalone blood cells in each anticoagulation protective agent after 1.5h in vitro (the group without anticoagulation protective agent is a control group);
FIG. 4 shows agglutination of abalone blood cells in each anticoagulation protective agent after 3h in vitro;
FIG. 5 shows agglutination of abalone blood cells in each anticoagulation protective agent after 4h ex vivo;
FIG. 6 shows agglutination of abalone blood cells in each anticoagulation protective agent after 5h ex vivo;
in FIGS. 3 to 6, the arrows indicate the phenomenon of cell aggregation.
Detailed Description
The invention will be further explained by means of specific embodiments, however, it should be understood that the invention may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
Example 1
The utility model provides a blood filter, as shown in fig. 1, 2, includes fixed station 1, montant 2, filter equipment 3 and puts thing device 4, it places in filter equipment 3's below to put thing device 4, filter equipment 3 installs on montant 2 through clamping ring pole 5, and clamping ring pole 5 passes through height adjusting knob 14 to be installed on montant 2, and montant 2 is installed on fixed station 1, filter equipment 3 includes clamping ring 6 and lower clamping ring 7, it all is connected with montant 2 with lower clamping ring 7 to go up clamping ring 6. A bolting silk is arranged between the upper pressing ring 6 and the lower pressing ring 7. The mesh 13 filters blood through a silk screen.
Furthermore, in order to facilitate the replacement of the bolting silk arranged between the upper pressing ring 6 and the lower pressing ring 7, four pressing buckles 8 are arranged on the upper pressing ring 6, and the four pressing buckles 8 are uniformly distributed on the periphery of the upper pressing ring 6. The four pressing buckles 8 can firmly buckle the upper pressing ring 6 on the lower pressing ring 7 and can buckle the bolting silk between the upper pressing ring 6 and the lower pressing ring 7.
Further, a grid mesh 13 is arranged on the upper pressing ring 6, and the grid mesh 13 is provided with four square grids. The bolting silk is divided into four parts by a grid 13.
Specifically, the storage device 4 comprises a storage box 9 and a centrifuge tube 10, and the centrifuge tube 10 is placed in the storage box 9. Centrifuge tube 10 is used to hold filtered blood.
Further, a cylindrical groove 11 is formed in the storage box 9, and ice cubes are placed in the cylindrical groove 11. And maintaining the low-temperature environment required by the filtered blood by using ice cubes.
Furthermore, two spacers 12 are disposed in the cylindrical groove 11, and the two spacers 12 are perpendicularly crossed. Through two spacers 12 of vertical cross arrangement, with four microgrooves of cylindric recess 11 split, four microgrooves and the square net one-to-one of grid 13 place a centrifuging tube 10 in every microgroove to be convenient for mark sample, can not cause and obscure.
Specifically, the material of the press button 8 is plastic. The upper pressing ring 6 and the lower pressing ring 7 are both made of stainless steel. The fixed table 1 is a diamond iron fixed table. The iron fixed station of rhombus can guarantee the utility model discloses a steadiness can not be emptyd because of being located 2 one sides of montant putting thing device 4.
When the bolting silk is arranged between the upper pressing ring 6 and the lower pressing ring 7, the bolting silk is divided into four parts by the grid mesh 13, a syringe or a pipette with a needle removed is used for sucking blood, the height adjusting button 14 is adjusted to be just aligned with four centrifuge tubes 10 of the storage box 9 (in which ice is put), one part of the bolting silk is aligned for downward injection, and the liquid enters the centrifuge tube 10 in the storage box 9 which is just opposite to the lower part through the bolting silk; each part of the bolting-silk is used once, when the bolting-silk in the ring is used up, the press button 8 is opened, and a brand-new bolting-silk is replaced. Through the blood filtered by the blood filter, impurities in the blood can be filtered, the purity of the blood is improved, and a regular and ordered cell scatter diagram aggregated into clusters can be obtained after the blood is detected by a flow cytometer.
Example 2
A method for processing abalone blood cells, comprising the following steps:
(1) firstly, adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube;
(2) then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily;
(3) after the blood flows out, 1ml of blood is pumped by a sterile syringe and is directly injected into a centrifuge tube added with the anticoagulation protective agent;
(4) filtering the liquid in step (3) into a new 2ml centrifuge tube using the hemofilter of example 1;
(5) centrifuging at 18 deg.C and 3000rpm for 10min, and removing supernatant;
(6) and (4) finally, resuspending the blood cells treated in the step (5) by using 1ml of filtered seawater to prepare abalone hemolymph suspension.
Wherein, the anticoagulant protective agent consists of the following components: 2.5g of sodium chloride, 1g of EDTA, 0.23g of 1, 4-piperazine-diethylsulphonic acid and 100ml of 0.1mM PBS.
The preparation method of the abalone blood cell anticoagulant protective agent specifically comprises the following steps:
(1) pouring 100ml of 0.1mM PBS buffer solution into a beaker, adding 2.5g of solid sodium chloride powder into the beaker, stirring for 3 minutes until the sodium chloride is completely dissolved, and then adding 0.23g of 1, 4-piperazine diethylsulfonic acid powder, fully stirring and dissolving to obtain a mixed solution A;
(2) then, putting the mixed solution A into a water bath kettle at 40 ℃ for heating, slowly adding 1g of EDTA (ethylene diamine tetraacetic acid) while stirring until the EDTA is completely dissolved to obtain a mixed solution B;
(3) the mixed solution B is stirred for 0.5h by a magnetic stirrer at room temperature, so that all components are fully dissolved in PBS buffer solution;
(4) and (3) starting an ultraviolet sterilization function for 30min on the ultra-clean workbench, filtering and removing impurities of the mixed solution B by using a 0.45um sterile filter on the ultra-clean workbench, putting the mixed solution B into a sterilized blue-cap bottle, filtering and sterilizing liquid in the blue-cap bottle by using a 0.22um sterile filter, and then obtaining the abalone blood cell anticoagulation protective agent.
Example 3
A method for processing abalone blood cells, comprising the following steps:
(1) firstly, adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube;
(2) then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily;
(3) after the blood flows out, 1ml of blood is pumped by a sterile syringe and is directly injected into a centrifuge tube added with the anticoagulation protective agent;
(4) filtering the liquid in step (3) into a new 2ml centrifuge tube using the hemofilter of example 1;
(5) centrifuging at 18 deg.C and 3000rpm for 10min, and removing supernatant;
(6) and (4) finally, resuspending the blood cells treated in the step (5) by using 1ml of filtered seawater to prepare abalone hemolymph suspension.
The anticoagulant protective agent consists of the following components: 2.55g of sodium chloride, 1.1g of EDTA, 0.24g of 1, 4-piperazine-diethylsulphonic acid and 100ml of 0.1mM PBS.
The preparation method of the abalone blood cell anticoagulant protective agent specifically comprises the following steps:
(1) pouring 100ml of 0.1mM PBS buffer solution into a beaker, adding 2.55g of solid sodium chloride powder into the beaker, stirring for 4 minutes until the sodium chloride is completely dissolved, and then adding 0.24g of 1, 4-piperazine diethylsulfonic acid powder, fully stirring and dissolving to obtain a mixed solution A;
(2) then, the mixed solution A is put into a water bath kettle at the temperature of 39 ℃ for heating, and then 1.1g of EDTA is slowly added while stirring until the EDTA is completely dissolved to obtain a mixed solution B;
(3) transferring the mixed solution B to a room temperature state, and stirring for 45min by using a magnetic stirrer to fully dissolve each component into the PBS buffer solution;
(4) and (3) starting an ultraviolet sterilization function of the ultra-clean workbench for 32min, then filtering and removing impurities of the mixed solution B by using a 0.45um sterile filter on the ultra-clean workbench, putting the mixed solution B into a sterilized blue-cap bottle, filtering and sterilizing liquid in the blue-cap bottle by using a 0.22um sterile filter, and then obtaining the abalone blood cell anticoagulation protective agent.
Example 4
A method for processing abalone blood cells, comprising the following steps:
(1) firstly, adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube;
(2) then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily;
(3) after the blood flows out, 1ml of blood is pumped by a sterile syringe and is directly injected into a centrifuge tube added with the anticoagulation protective agent;
(4) filtering the liquid in step (3) into a new 2ml centrifuge tube using the hemofilter of example 1;
(5) centrifuging at 18 deg.C and 3000rpm for 10min, and removing supernatant;
(6) and (4) finally, resuspending the blood cells treated in the step (5) by using 1ml of filtered seawater to prepare abalone hemolymph suspension.
The anticoagulant protective agent consists of the following components: 2.45g of sodium chloride, EDTA0.9g, 0.22g of 1, 4-piperazine disulfonic acid and 100ml of 0.1mM PBS.
The preparation method of the abalone blood cell anticoagulant protective agent specifically comprises the following steps:
(1) pouring 100ml of 0.1mM PBS buffer solution into a beaker, adding 2.45g of solid sodium chloride powder into the beaker, stirring for 5 minutes until the sodium chloride is completely dissolved, and then adding 0.22g of 1, 4-piperazine diethylsulfonic acid powder, fully stirring and dissolving to obtain a mixed solution A;
(2) heating the mixed solution A in a 38 ℃ water bath, slowly adding 0.9g of EDTA while stirring until the EDTA is completely dissolved to obtain a mixed solution B;
(3) the mixed solution B is stirred for 1 hour by a magnetic stirrer at room temperature, so that all components are fully dissolved in PBS buffer solution;
(4) the ultra-clean workbench is started to have an ultraviolet sterilization function for 35min, then the mixed solution B is filtered and decontaminated by a 0.45um sterile filter on the ultra-clean workbench and is put into a sterilized blue-cap bottle, then the liquid in the blue-cap bottle is filtered and sterilized by a 0.22um sterile filter, and then the obtained abalone blood cell anticoagulation protective agent is obtained.
To further prove the beneficial effects of the present invention, the applicant also carried out the following tests:
comparison of anticoagulation activities of different anticoagulation protective agents
The formula of the No. 1 anticoagulation protective agent (the formula of the anticoagulation protective agent is recorded as AP): 2.5g of sodium chloride, 100ml of 0.1mM PBS, 1g of EDTA and 0.23g of 1, 4-piperazine-diethyl sulfonic acid;
the formula of the No. 2 anticoagulant protective agent is as follows: 2.5g of sodium chloride, 100ml of 0.1mM PBS and 1g of EDTA;
the formula of the No. 3 anticoagulant protective agent is as follows: 2.5g of sodium chloride, 100ml of 0.1mM PBS, 1g of EDTA and 0.15g of sodium oxalate.
Firstly, mixing abalone blood cells and different anticoagulation protective agents (No. 1, 2 and 3 anticoagulation protective agents) according to the proportion of 1: the agglutination of the blood cells after 1 volume mixing and 1.5h at room temperature is shown in Table 1 and FIG. 3, and pure abalone blood cells without the anticoagulation protective agent are added as a control in this comparison.
TABLE 1 agglutination of abalone blood cells in different anticoagulation protective agents
Note: the number of clumps is the average number of cell clumps per field of view among 20 fields observed at random.
Under the observation of a microscope, abalone blood cells without the addition of the anticoagulation protective agent are aggregated, a plurality of cell aggregation groups are formed, and the cell aggregation number of each aggregation group is more than 20 cells. Abalone blood cells are uniformly distributed in AP, the number of blood cell clusters in 20 randomly observed visual fields is less than 2, the number of blood cells in each cell cluster is less than 3, and the anti-aggregation effect is best. The second effect of the No. 2 anticoagulation protective agent is that the hemagglutination phenomenon in the No. 3 anticoagulation protective agent generally occurs.
On the basis of the above experiment, the anticoagulation condition of the abalone blood cells added with the number 1, 2 and 3 anticoagulation protective agents is continuously observed. The agglutination of the blood cells after 3h, 4h and 5h at the number 1, 2 and 3 anticoagulation protective agents is compared at room temperature, and the specific comparison results are shown in Table 2 and FIGS. 4-6.
TABLE 2 coagulation of abalone blood cells in different anticoagulant protectors at different time periods
As can be seen from FIG. 4, the blood cells were uniformly distributed in AP after 3 hours, and the blood cells in the visual field tended to agglutinate, and the number of agglutinated cells was only 3-4. The blood cells are unevenly distributed in the No. 2 anticoagulation protective agent, the blood cells in a visual field have obvious agglutination phenomenon, the number of the agglutinated cells exceeds 5, and the phenomenon of flocculent impurities occurs. The blood cells are unevenly distributed in the No. 3 anticoagulation protective agent, the blood cells in a visual field have obvious agglutination phenomenon, and the number of the agglutinated cells exceeds 10.
As can be seen from FIG. 5, the distribution of blood cells in AP was uniform after 4 hours, and a small amount of cell aggregates appeared in the visual field, but the number of aggregated cells did not exceed 4, and the aggregation phenomenon was not serious. The blood cells are unevenly distributed in the anticoagulant protective agent No. 2, a cell agglutination group is obvious in a visual field, and the number of agglutinated cells exceeds 8. The distribution of blood cells in the anticoagulant protective agent No. 3 is very uneven, the number of cell agglutinates is more in visual field, and the number of agglutinated cells exceeds 20.
As can be seen from FIG. 6, the distribution of blood cells in AP was less uniform after 5 hours, and a small amount of cell aggregates appeared in the visual field, but the number of aggregated cells did not exceed 6, and the aggregation phenomenon was not serious. As can be seen, after 5 hours, the blood cells are unevenly distributed in the anticoagulant protectant No. 2, a large amount of cell aggregates appear in the visual field, and the aggregation phenomenon is serious. After 5 hours, the blood cells are not uniformly distributed in the No. 3 anticoagulation protective agent, the cell coagulation groups are more in the visual field, the number of the coagulated cells is more (more than 22), and the coagulation phenomenon is serious.
Tests prove that the AP anticoagulation protective agent is obviously superior to the anticoagulation protective agents 2 and 3 in the aspects of maintaining cell morphology and preventing cell agglutination in different time periods (0-5h), and is an ideal choice in research of abalone tests.
Second, comparison of the cell death rates of different anticoagulant protectors
The determination of the blood cell death rate was carried out using a flow cytometer. The specific operation steps are as follows: 500ul abalone blood (after adding different anticoagulation protective agents) is pumped into a new 2ml sterilized centrifuge tube, 10ul PI dye solution (with the final concentration of 20ug/m1) is respectively added, and incubation is carried out for 10min in a dark room at the temperature of 20 ℃. Centrifugation at 400g for 5min at 20 ℃ removed unstained PI reagent. The cells were resuspended in 600ul of sterile seawater and placed in a 5ml flow tube. The cells were counted for 30 seconds using a flow cytometer. The fluorescent signal at 630nm was obtained, and the dead cells were distinguished from the live cells by a single parameter histogram of the FL2 channel of the flow cytometer.
The results of the cell death rates measured after equal volumes of abalone blood cells and various anticoagulant protectors were mixed are shown in Table 3.
TABLE 3 mortality of blood cells in each anticoagulation protectant
As can be seen from Table 3, blood cells in AP showed lower mortality rates in 1.5-5h, while anticoagulants No. 2 and No. 3 showed higher mortality rates than AP. In combination with the anticoagulation effects of tables 1 and 2, the AP anticoagulation protective agent of the invention is more effective.
Third, the treatment effect of the blood filter of the invention
Compared with the effects before and after filtration, the hemolymph is filtered by the hemofilter, a large amount of impurities are removed, the cleaning time of the flow cytometer is shortened, the analysis error is reduced, and the obtained experimental result is more accurate.
And the time and cost for cleaning the abalone blood cell sample on the machine are compared, and the specific comparison result is shown in table 4.
TABLE 4 abalone blood cell samples cleaning time and cost on machine
To examine the effect of the cell treatment, the applicant proceeded with the following examination, in particular: the phagocytic activity of blood cells was measured by a high-precision instrument such as a flow cytometer. The method comprises the following specific steps: 500ul abalone hemolymph suspension is put into a 2ml centrifuge tube, 88ul fluorescent microsphere liquid (10 ul fluorescent microsphere and 490ul filtered seawater → 2% fluorescent microsphere liquid) is added, and the mixture is evenly mixed and then placed in a dark room at 20 ℃ for incubation for 60 minutes. 300ul of cold anticoagulant protector was added to each centrifuge tube to stop the reaction. Centrifuge at 400g for 5min at 20 deg.C (the uncophagocytosed fluorescent microspheres are in the supernatant) and discard the supernatant. The cells were resuspended in 600ul of filtered seawater and the sample was poured into a 5ml flow cytometer for assay, the specific assay results are shown in table 5.
TABLE 5 Effect of cell treatment on phagocytic Activity of cells
As can be seen from table 5, the phagocytic activity values of the cells without the anticoagulation protective agent and filter treatment were significantly lower than those of the treated group. This indicates that untreated cells significantly affect phagocytic activity and ultimately the accuracy of the experimental assay. The treated cells do not influence the phagocytic activity of the cells, and the accurate measurement value can be obtained in the experiment.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. An abalone blood cell processing method, characterized in that the processing method comprises the following steps:
(1) firstly, adding 1ml of anticoagulation protective agent into a 2ml centrifuge tube;
(2) then, cutting open the blood sinuses of the abdominal foot of the abalone by using a scalpel, and putting the abalone on ice with an inclination of 20-30 degrees so that blood can flow out more easily; (3) after the blood flows out, pumping 1ml of blood by using a sterile syringe and directly injecting the blood into a centrifuge tube added with the anticoagulation protective agent; the anticoagulant protective agent comprises the following components: 2.45-2.55 g of sodium chloride, 0.9-1.1 g of EDTA, 0.22-0.24 g of 1, 4-piperazine diethyl sulfonic acid and 100ml of 0.1mM PBS;
(4) filtering the liquid of step (3) with a hemofilter for flow cytometry into a new 2ml centrifuge tube; (5) centrifuging at 18 ℃ and 3000rpm for 10min, and removing the supernatant;
(6) and (4) finally, resuspending the blood cells treated in the step (5) by using 1ml of filtered seawater to prepare abalone hemolymph suspension.
2. The abalone blood cell processing method according to claim 1, wherein the anticoagulation protecting agent is prepared by:
(1) pouring 0.1mM PBS buffer solution into a beaker, adding solid sodium chloride powder into the beaker, stirring for 3-5 minutes until the sodium chloride is completely dissolved, then adding 1, 4-piperazine diethylsulfonic acid powder, and fully stirring and dissolving to obtain a mixed solution A; (2) heating the mixed solution A in a 38-40 ℃ water bath, slowly adding EDTA (ethylene diamine tetraacetic acid) while stirring until the EDTA is completely dissolved to obtain a mixed solution B;
(3) the mixed solution B is stirred for 0.5 to 1 hour by a magnetic stirrer at room temperature, so that all the components are fully dissolved in PBS buffer solution;
(4) and (3) starting an ultraviolet sterilization function of the ultra-clean workbench for 30-35min, then filtering and removing impurities of the mixed solution B on the ultra-clean workbench by using a 0.45um sterile filter, putting the mixed solution B into a sterilized blue-cap bottle, and filtering and sterilizing the liquid in the blue-cap bottle by using a 0.22um sterile filter to finally obtain the abalone blood cell anticoagulant protective agent.
3. The abalone blood cell processing method according to claim 1, characterized in that the blood filter comprises a fixed platform (1), a vertical rod (2), a filtering device (3) and an object placing device (4), the object placing device (4) is placed below the filtering device (3), the filtering device (3) is installed on the vertical rod (2) through a pressing ring rod (5), the pressing ring rod (5) is installed on the vertical rod (2) through a height adjusting button (14), the vertical rod (2) is installed on the fixed platform (1), the filtering device (3) comprises an upper pressing ring (6) and a lower pressing ring (7), the upper pressing ring (6) and the lower pressing ring (7) are both connected with the pressing ring rod (5), and a bolting silk is arranged between the upper pressing ring (6) and the lower pressing ring (7).
4. An abalone blood cell processing method according to claim 3, characterised in that the upper clamping ring (6) is provided with four press buttons (8) which are evenly distributed around the upper clamping ring (6).
5. An abalone blood cell processing method according to claim 4, characterized in that the upper pressing ring (6) is provided with a grid mesh (13), and the grid mesh (13) has four square grids.
6. An abalone blood cell processing method according to claim 3, characterized in that the storage device (4) comprises a storage box (9) and a centrifuge tube (10), the centrifuge tube (10) being placed in the storage box (9).
7. An abalone blood cell processing method according to claim 6, characterised in that the storage box (9) is provided with a cylindrical groove (11), and ice cubes are placed in the cylindrical groove (11).
8. An abalone blood cell processing method according to claim 7, characterized in that two spacers (12) are provided in the cylindrical recess (11), and the two spacers (12) are arranged to intersect vertically.
9. The abalone blood cell processing method according to claim 4, wherein the press button (8) is made of plastic, the upper press ring (6) and the lower press ring (7) are both made of stainless steel, and the fixing table (1) is a diamond iron fixing table.
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