CN115125185A - Preparation method of bivalve blood lymphocyte single cell suspension - Google Patents
Preparation method of bivalve blood lymphocyte single cell suspension Download PDFInfo
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Abstract
The invention discloses a preparation method of bivalve hemolymph cell single cell suspension. The preparation method comprises the following steps: selecting bivalve shellfish and collecting hemolymph thereof; measuring the osmotic pressure of plasma in the haemolymph, and adjusting the osmotic pressure of the buffer solution to ensure that the osmotic pressure of the plasma is the same as that of the buffer solution; centrifuging the collected hemolymph, and collecting a cell precipitate I; adding a buffer solution into the cell sediment, and performing suction to resuspend the cells to obtain a cell mixed solution I; centrifuging the cell mixed solution I, and collecting a cell precipitate II; repeating the cell precipitation to obtain a cell mixed solution II; centrifuging the cell mixed solution II, and collecting a cell precipitate III; repeating the cell precipitation for more than three times to obtain a cell mixed solution III; and filtering the cell mixed solution III by using a cell filter to finally obtain the bivalve hemolymph cell single cell suspension. The method does not need to prepare the haemolymph anticoagulant, and can quickly and efficiently obtain the bivalve haemolymph single cell suspension with low cost.
Description
Technical Field
The invention belongs to the technical field of marine invertebrate cell biology, and relates to a preparation method of a bivalve hemolymphocyte single cell suspension.
Background
The bivalve shellfish has an open circulatory system, and mainly depends on blood lymph circulation to realize various physiological functions, such as gas exchange, nutrient substance transportation, cellular immunity, secretion excretion, etc. The bivalve hemolymph consists of hemolymph fluid and hemolymph cells, wherein the hemolymph cells are the main immune cells of the bivalve and participate in the physiological processes of phagocytosis, encystment, antibacterial peptide synthesis and the like. Blood lymphocytes comprise a variety of cell types with structural and functional heterogeneity, with different types of blood lymphocytes performing different functions in the immune system. However, the classification of bivalve hemolymphocytes is mostly limited at the morphological level at present, and the characterization of different cell types at the molecular level is not realized yet.
The emergence of the single cell sequencing technology enables researchers to classify cells according to the gene expression condition in the single cells, analyze the cell state, identify rare cell types, infer cell differentiation approaches and the like, and provides an effective technical means for deeply analyzing the blood lymphocyte functional heterogeneity of the bivalve shellfish. The preparation of high-quality single-cell suspension is the key of single-cell transcriptome sequencing, and a high-throughput single-cell sequencing instrument requires cell flow sorting, but in the actual experimental process, the requirements of on-machine library construction cannot be met due to the problems of insufficient cell amount, poor cell activity, cell breakage, agglomeration and the like in the cell suspension.
Therefore, it is necessary to develop a preparation method suitable for the bivalve hemolymph cell single cell suspension, which is simple to operate, can efficiently and quickly dissociate the bivalve hemolymph cells and ensures good cell activity.
Disclosure of Invention
The invention aims to provide a preparation method of bivalve hemolymph cell single cell suspension to make up for the defects of the prior art.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
a preparation method of bivalve hemolymph single cell suspension comprises the following steps:
(1) selecting bivalve shellfish and collecting hemolymph thereof;
(2) measuring the osmotic pressure of plasma in the haemolymph, and adjusting the osmotic pressure of the buffer solution to ensure that the osmotic pressure of the plasma is the same as that of the buffer solution;
(3) centrifuging the collected hemolymph at the temperature of 4 ℃ and the temperature of 600-700 g for 5-6 min, removing supernatant, and collecting cell precipitate I;
(4) adding a buffer solution pre-cooled at 4 ℃ into the cell sediment, and performing suction to resuspend the cells to obtain a cell mixed solution I;
(5) centrifuging the cell mixed solution I at the temperature of 4 ℃ for 5-6 min at 600-700 g, removing supernatant, and collecting cell precipitate II;
(6) repeating the step (4) on the cell sediment to obtain a cell mixed solution II;
(7) centrifuging the cell mixed solution II at the temperature of 4 ℃ for 3-5 min at 300-400 g, removing supernatant, and collecting cell precipitate III;
(8) repeating the step (4) for three times to obtain a cell mixed solution III;
(9) and filtering the cell mixed solution III by using a cell filter to finally obtain the bivalve hemolymph cell single cell suspension.
Further, the step (1) is specifically as follows: selecting bivalve shellfish with good activity, slightly cutting a small opening on the adductor muscle of bivalve shellfish by using a blade, and collecting hemolymph flowing out of the adductor muscle by using a syringe after the hemolymph naturally flows out; in addition, the pericardial cavity is cut open and the hemolymph in the pericardial cavity is collected.
Further, in the step (1), the collected hemolymph is placed on ice to prevent coagulation.
Further, the buffer solution was 0.0265M PBS buffer, and the solution was isotonic with the plasma osmotic pressure of bivalve shellfish.
Further, the pore size of the cell filter is 40 μm.
The bivalve hemolymph cell single cell suspension obtained by the method can be used for subsequent detection and analysis: and sucking the cell suspension, staining the cell suspension by using a 0.2-0.4% trypan blue solution, and detecting the concentration, activity and agglomeration condition of the single cells by using a cell counting plate under a microscope.
The purpose of reducing the centrifugal force and the centrifugation time in the above-described method is to help reduce cell debris and impurities in the single cell suspension compared to the prior art.
Compared with the prior art, the invention has the advantages that:
the method for preparing the bivalve hemolymph cell single cell suspension does not need to be provided with a hemolymph anticoagulant, can realize low-cost, quick and efficient obtaining of the bivalve hemolymph cell single cell suspension, can ensure that the hemolymph cells obtained by dissociation keep good activity, has few impurities, reduces cell fragmentation, aggregation and the like to the maximum extent, and ensures the concentration of living cells and the quality of the single cell suspension.
The invention can be applied to the separation and sorting of bivalve hemolymphocytes, the in vitro culture of cells and the establishment of a cell line, simultaneously provides reference for the dissociation of other marine invertebrate cells, and provides important reference data for genetic breeding researches such as bivalve unicell sequencing, cell type identification, cell function research and the like.
Drawings
FIG. 1 is a first view of a patinopecten yessoensis blood lymphocyte suspension used for single cell sequencing under an optical microscope according to an embodiment of the present invention.
Fig. 2 is a second image of the patinopecten yessoensis blood lymphocyte suspension used for single cell sequencing under an optical microscope according to the embodiment of the present invention.
FIG. 3 is a view of a suspension of patinopecten yessoensis blood lymphocytes for single cell sequencing under an optical microscope according to a comparative example of the present invention.
FIG. 4 is a view of a suspension of patinopecten yessoensis blood lymphocytes for single cell sequencing under an optical microscope according to a comparative example II of the present invention.
Detailed Description
The present invention will be described in further detail to explain the technical solutions of the present invention more clearly. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Example 1 a specific experiment was conducted by taking Japanese scallop as an example.
A method for preparing a single cell suspension of Japanese scallop blood lymphocytes comprises the following steps:
1) selecting Japanese scallop with good activity, slightly scratching a small opening on adductor muscle of the Japanese scallop by using a surgical blade, collecting hemolymph flowing out of the adductor muscle by using a 5mL disposable syringe after the hemolymph naturally flows out, and additionally scratching a pericardial cavity and collecting the hemolymph in the pericardial cavity;
2) transferring the collected hemolymph into a sterile 2mL centrifuge tube, and placing on ice to prevent the hemolymph from agglutinating;
3) measuring the osmotic pressure of plasma in the hemolymph, and adjusting the osmotic pressure of the PBS buffer solution to ensure that the osmotic pressure of the plasma is the same as that of the PBS buffer solution, wherein the finally measured concentration of the PBS buffer solution is 0.0265M;
4) centrifuging the hemolymph of the step 2) at the temperature of 4 ℃ and the temperature of 650g for 5min, removing supernatant, and collecting the hemolymph cell sediment I accumulated at the bottom of the tube;
5) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the first sediment, gently sucking the first sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a first cell mixed solution;
6) centrifuging the first cell mixed solution obtained in the step 5) at 4 ℃ and 650g for 5min, removing supernatant, and collecting a second hemolymphocyte precipitate accumulated at the bottom of a tube;
7) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the second sediment, gently blowing and sucking for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a second cell mixed solution;
8) centrifuging the cell mixed solution II obtained in the step 7) at 4 ℃ under 300g for 4min, removing supernatant, and collecting the hemolymph cell sediment III accumulated at the bottom of the tube;
9) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the third sediment, gently sucking the third sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a hemolymph cell suspension;
10) filtering the blood lymphocyte suspension obtained in the step 9) by using a cell filter of 40 mu m to obtain a comb shell blood lymphocyte single cell suspension suitable for single cell sequencing;
11) performing 10 x Genomics single cell sequencing on the patinopecten yessoensis hemolymph single cell suspension obtained in the step 10).
The results show that:
10. mu.L of the single cell suspension prepared above was aspirated, stained with 0.4% Trypan blue solution, and the concentration, activity and aggregation of single cells were examined under a microscope using a cell counting plate.
As shown in fig. 1 and 2, the background debris is less, the cells are clearly visible, the morphology is good, and almost no agglomeration occurs; the cell concentration of the comb shell blood lymphocyte single-cell suspension is about 1200 cells/microliter, the cell survival rate is 93 percent, no cell mass or other impurities larger than 40 mu m exist, the cell agglomeration rate is about 0.01 percent, and the cell agglomeration rate can meet the requirement of 10 x Genomics single-cell sequencing machine.
As shown in Table 1, the single cell suspension of Japanese scallop blood lymphocytes prepared in example 1 was subjected to in-situ library sequencing, total sequencing cell number 10145, and total basis number 13778 were co-monitored. The total reads number of sequencing is 214762962, the effective Barcodes proportion is 96.3 percent, the Barcodes sequence is more than 94.3 percent of Q30, the RNA sequence is more than 92.5 percent of Q30, and the UMI sequence is more than 93.6 percent of Q30. The comparison data with the reference genome shows that the ratio of reads on the reference genome is 96.3%, and the ratio of reads on the reference genome is 87%, wherein the ratios of reliable comparison to intergenic region, intronic region and exonic region are respectively: 25.9%, 5.1% and 56%. The quality of the sample data is high, and the reliability of subsequent analysis can be ensured.
TABLE 1 quality test report of construction of Patinopecten yessoensis hemolymphocyte single-cell transcriptome library
Comparative example 1:
the osmotic pressure of the buffer was adjusted in comparative example 1 as compared with example 1.
A method for preparing single cell suspension of Japanese scallop blood lymphocytes comprises the following steps:
1) selecting Japanese scallop with good activity, slightly scratching a small opening on adductor muscle of the Japanese scallop by using a surgical blade, collecting hemolymph flowing out of the adductor muscle by using a 5mL disposable syringe after the hemolymph naturally flows out, and additionally scratching a pericardial cavity and collecting the hemolymph in the pericardial cavity;
2) transferring the collected hemolymph into a sterile 2mL centrifuge tube, and placing on ice to prevent the hemolymph from agglutinating;
3) centrifuging the hemolymph of the step 2) at the temperature of 4 ℃ and the temperature of 650g for 5min, removing supernatant, and collecting the hemolymph cell sediment I accumulated at the bottom of the tube;
4) adding 1.5mL of 0.01M PBS buffer solution precooled at 4 ℃ into the first sediment, gently sucking the first sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a first cell mixed solution;
5) centrifuging the first cell mixed solution obtained in the step 4) at 4 ℃ and 650g for 5min, removing supernatant, and collecting a second hemolymphocyte precipitate accumulated at the bottom of a tube;
6) adding 1.5mL of 0.01M PBS buffer solution precooled at 4 ℃ into the second sediment, gently sucking the second sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a second cell mixed solution;
7) centrifuging the cell mixed solution II obtained in the step 6) for 4min at the temperature of 4 ℃ and the speed of 300g, removing supernatant, and collecting a hemolymph cell precipitate III accumulated at the bottom of a tube;
8) adding 1.5mL of 0.01M PBS buffer solution precooled at 4 ℃ into the third sediment, gently sucking the third sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a hemolymph cell suspension;
9) filtering the blood lymphocyte suspension obtained in the step 8) by using a cell filter of 40 mu m to obtain the comb shell blood lymphocyte single cell suspension suitable for single cell sequencing.
The results show that:
10. mu.L of the single cell suspension prepared above was aspirated, stained with 0.4% Trypan blue solution, and the concentration, activity and aggregation of single cells were examined under a microscope using a cell counting plate.
The results show that the survival rate of the single cell suspension of patinopecten yessoensis hemolymph cells is lower, less than 70%, as shown in fig. 3, although there is no cell mass or other impurities larger than 40 μm, the survival rate has higher failure risk for 10 x Genomics single cell sequencing machine.
Comparative example 2:
in comparative example 2, the centrifugal force and the centrifugal time in step 8) were adjusted as compared with example 1.
A method for preparing a single cell suspension of Japanese scallop blood lymphocytes comprises the following steps:
1) selecting Patinopecten yessoensis with good activity, slightly cutting a small opening on the adductor muscle of the Patinopecten yessoensis by using a surgical blade, collecting hemolymph flowing out of the adductor muscle by using a 5mL disposable syringe after the hemolymph naturally emerges, and cutting a pericardial cavity to collect the hemolymph in the pericardial cavity;
2) transferring the collected hemolymph into a sterile 2mL centrifuge tube, and placing on ice to prevent the hemolymph from agglutinating;
3) measuring the plasma osmotic pressure in the hemolymph single cell, and adjusting the osmotic pressure of the PBS buffer solution to ensure that the osmotic pressure of the hemolymph single cell and the osmotic pressure of the PBS buffer solution are the same, wherein the finally measured concentration of the PBS buffer solution is 0.0265M;
4) centrifuging the hemolymph of the step 2) at the temperature of 4 ℃ and the temperature of 650g for 5min, removing supernatant, and collecting the hemolymph cell sediment I accumulated at the bottom of the tube;
5) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the first sediment, gently sucking the first sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a first cell mixed solution;
6) centrifuging the first cell mixed solution obtained in the step 5) at 4 ℃ and 650g for 5min, removing supernatant, and collecting a second hemolymphocyte precipitate accumulated at the bottom of a tube;
7) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the second sediment, gently blowing and sucking for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a second cell mixed solution;
8) centrifuging the cell mixed solution II obtained in the step 7) for 5min at the temperature of 4 ℃ and the temperature of 650g, removing supernatant, and collecting a hemolymph cell precipitate III accumulated at the bottom of a tube;
9) adding 1.5mL of 0.0265M PBS buffer solution precooled at 4 ℃ into the third sediment, gently sucking the third sediment for 3-5 times by using a 1mL sterile suction head, and re-suspending the cells to obtain a hemolymph cell suspension;
10) filtering the blood lymphocyte suspension obtained in the step 9) by using a cell filter of 40 mu m to obtain the comb shell blood lymphocyte single cell suspension suitable for single cell sequencing.
The results show that:
10. mu.L of the single cell suspension prepared above was aspirated, stained with 0.4% Trypan blue solution, and the concentration, activity and aggregation of single cells were examined under a microscope using a cell counting plate.
The results show that the survival rate of the cells in the single cell suspension of patinopecten yessoensis hemolymphocytes is high, and the survival rate is not obviously different from the examples, and the cell agglomeration proportion is higher (as shown in figure 4), but the prepared single cell suspension has more cell fragments and impurities, the fragment proportion is higher, and the single cell suspension has higher failure risk on a 10 x Genomics single cell sequencer.
According to the experimental results provided by the invention in the embodiment 1 of the invention, it is demonstrated that the single cell suspension of patinopecten yessoensis blood lymphocytes prepared by the technical scheme provided by the invention can meet the quality index requirements of a single cell sequencing machine, and smoothly complete the single cell sequencing of the patinopecten yessoensis blood lymphocytes.
In order to develop a single cell suspension preparation method suitable for the single cell sequencing specificity of bivalve hemolymph cells, the invention carries out a comparative experiment. The focus of the present invention is on the exploration of the osmotic pressure of the cell buffer solution and how to remove the cell impurities and debris simply and efficiently, and according to the operation sequence of the process, the osmotic pressure of the cell buffer solution is firstly explored and determined, as described in comparative example 1; next, the centrifugal force and time for separating the cell pellet were compared as described in comparative example 2.
Comparative example 1 the experiment was performed using the most common 0.01M PBS solution as the cell buffer solution, and as shown in comparative example 1, compared with example 1, the single cell suspension of blood lymphocytes prepared in comparative example 1 has lower cell concentration and lower cell viability, and cannot meet the cell quantity requirement of the on-machine banking.
Comparative example 2 the centrifugal force and time in step 8) of example 1 were changed, and the cell pellet was further washed with 300g for 4min, as shown in comparative example 2, compared with example 1, the amount of the single cell suspension of haemolymph cells prepared in comparative example 2 was not significantly different, but more cell debris and impurities were observed under an optical microscope, which failed to meet the requirement of on-machine library construction.
The method is beneficial to efficiently and quickly dissociating the bivalve hemolymph cells, ensures that the dissociated cells keep good cell morphology and activity, prepares high-quality single cell suspension, and has important application value for the separation and classification of the bivalve hemolymph cells, the subsequent single cell sequencing, the cell function analysis and the establishment of a cell line.
Claims (4)
1. A preparation method of bivalve hemolymph single cell suspension is characterized by comprising the following steps:
(1) selecting bivalve shellfish and collecting hemolymph thereof;
(2) measuring the osmotic pressure of plasma in the haemolymph, and adjusting the osmotic pressure of the buffer solution to ensure that the osmotic pressure of the plasma is the same as that of the buffer solution;
(3) centrifuging the collected hemolymph at the temperature of 4 ℃ and the temperature of 600-700 g for 5-6 min, removing supernatant, and collecting cell precipitate I;
(4) adding a buffer solution pre-cooled at 4 ℃ into the cell sediment, and performing suction to resuspend the cells to obtain a cell mixed solution I;
(5) centrifuging the cell mixed solution I at the temperature of 4 ℃ for 5-6 min at 600-700 g, removing supernatant, and collecting cell precipitate II;
(6) repeating the step (4) on the cell sediment to obtain a cell mixed solution II;
(7) centrifuging the cell mixed solution II at the temperature of 4 ℃ for 3-5 min at 300-400 g, removing supernatant, and collecting cell precipitate III;
(8) repeating the step (4) on the cell precipitation to obtain a cell mixed solution III;
(9) and filtering the cell mixed solution III by using a cell filter to finally obtain the bivalve hemolymph cell single cell suspension.
2. The method of claim 1, wherein in the step (1), the collected hemolymph is placed on ice to prevent coagulation.
3. The method of claim 1, wherein the buffer solution is 0.0265M PBS buffer, and the solution is isotonic with the plasma osmotic pressure of bivalve shellfish.
4. The method of claim 1, wherein the pore size of the cell filter is 40 μm.
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