Separation liquid and separation method for separating shark PBMC

Abstract

The present invention relates to a separating solution for separating shark PBMC, which contains 1-2 g of NaCl in 100 mL of Ficoll-Urografin layering solution, and the density of the separating solution is 1.087 g / mL - 1.097 g / mL. The separation method is to mix fresh anticoagulated whole shark blood with shark blood diluent, then combine it with the separating solution, and after centrifugation, a PBMC layer with obvious stratification is obtained. After multiple cell resuspensions and centrifugal separations, high-purity shark PBMC is obtained. The separating solution has simple components, the separation operation is simple and easy to perform, and each component is a very stable substance. The prepared separating solution is easy to store, which simplifies the preparation process and storage conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of biological immunology, and particularly relates to a separation liquid and a separation method for isolating shark PBMCs. Background Art

[0002] PBMC (peripheral blood mononuclear cell) refers to peripheral blood mononuclear cells, that is, cells with a single nucleus in the blood, including T / B lymphocytes, monocytes, phagocytes, dendritic cells and other small cell types, among which lymphocytes account for a large proportion. In addition to PBMCs in peripheral blood, there are also mature red blood cells, multinucleated granulocytes and anucleate platelets. PBMCs are a key component of the immune system and are widely used as the most basic experimental materials in the fields of immunology, malignant tumors, vaccine research and development, transplantation treatment, etc. Therefore, the isolation of peripheral blood PBMCs has become the basis for the study of immune cells. The specific gravity of peripheral blood PBMCs is about 1.070, while the specific gravity of red blood cells and multinucleated granulocytes exceeds 1.080. Therefore, density gradient centrifugation can be used to isolate PBMCs.

[0003] The Ficoll-Paque density gradient centrifugation method has become the main method for isolating PBMCs from peripheral blood in current laboratories due to its relatively low price and simple operation. In addition to Ficoll-Paque, another commonly used cell separation liquid medium is Percoll, which is mainly composed of silica gel particles coated with a single layer of vinylpyrrolidone (PVP) on the surface and can also be used to prepare a separation liquid for animal peripheral blood mononuclear cells. For example, Patent 202111035419.7 discloses a gradient centrifugation method using Percoll separation solutions with different densities to separate the components of diluted anticoagulated whole blood cells of tree shrews and obtain PBMCs.

[0004] The marbled bambooshark belongs to the order Orectolobiformes, family Orectolobidae, subclass Elasmobranchii, and is a small benthic cartilaginous fish living in the coastal areas of southern China. Currently, there are more and more reports on the domestic research and development of nanobodies using marbled bamboosharks, and the demand for isolating their PBMCs for the construction of nanobody libraries is increasing day by day. Like other sharks and rays, marbled bamboosharks belong to cartilaginous fish. The immune system of cartilaginous fish is very ancient, and some species even lack adaptive immunity; its hematopoietic system is very different from that of humans. For example, the red blood cells of cartilaginous fish have nuclei, but lack bone marrow and lymph nodes. Therefore, studying the hematopoietic system of sharks will help us better understand immunology from an evolutionary perspective. Because the cell dispersion coefficients and cell charges of different species are different, strictly speaking, the corresponding density PBMC separation liquid of the corresponding species should be used.

[0005] There are various densities available for the existing Ficoll-Urografin density gradient medium on the market, such as 1.073, 1.077, 1.084 g / mL, which are mainly used for separating mammalian peripheral blood PBMC. However, these density gradient media are not suitable for separating shark PBMC. If the above-mentioned density gradient medium is used to separate shark blood PBMC, the obtained PBMC layer is still adhered to the red blood cell layer, and PBMC without red blood cells cannot be obtained. Another method, which is also commonly used in the literature, is to directly centrifuge shark blood at low speed to separate PBMC, but the same problem will be encountered, that is, the PBMC layer is adhered to the red blood cell layer, and the yield of PBMC is very low.

[0006] As of the filing of this invention patent application, the only patent literature (application number 202210468678.7) related to the method for separating shark mononuclear cells reported a method for separating shark mononuclear cells, a shark diluent and its uses. This separating solution is based on the reagent for separating human peripheral blood mononuclear cells and added with certain concentrations of inorganic salts, urea, water, etc., reducing the density of the separating solution from 1.077 g / mL to between 1.049 - 1.058 g / mL, and the centrifugal force used in the separation step is 1000 g, so as to separate shark mononuclear cells from red blood cells and granulocytes. However, this method requires a relatively large dilution ratio of blood (2 - 3 times), and the ratio of the separating solution reagent to the shark diluent is relatively high (about between 6:4 and 7:3), so the reagent cost and time cost consumed are relatively high, and the centrifugal force is relatively large, which may have a certain impact on cell viability. Summary of the Invention

[0007] In view of this, a separating solution and a separation method for separating shark PBMC, which are easy to store, simple to operate and have high separation purity, are provided, effectively solving the problem that the existing commonly used Ficoll-Urografin density gradient medium cannot separate shark PBMC from red blood cells. The separated PBMC has high purity and large quantity, providing an effective solution for the demand of high-purity PBMC in shark nanobody and immunology research.

[0008] A separating solution for separating shark PBMC, wherein the separating solution contains 1 - 2 g of NaCl in 100 mL of Ficoll-Urografin density gradient medium, and the density of the separating solution is 1.087 g / mL - 1.097 g / mL.

[0009] Preferably, the density of the density gradient medium is 1.077 g / mL.

[0010] Preferably, the separating solution contains 1.25 - 1.75 g of NaCl in 100 mL of Ficoll-Urografin density gradient medium, and the density of the separating solution is 1.090 g / mL - 1.095 g / mL. By adding NaCl, the density, osmotic pressure, etc. of the liquid can be accurately changed.

[0011] And, a separation method for separating shark PBMC, which comprises the following steps:

[0012] a. After mixing fresh anticoagulated whole shark blood with shark blood diluent, add them to a centrifuge tube in a predetermined volume ratio with the separation liquid for separating shark PBMC as described above. First add the separation liquid, and then add the diluted shark blood, so that the two liquids are fixedly separated into upper and lower layers without mixing. The upper layer is the diluted shark blood, and the lower layer is the separation liquid, maintaining a clear interface between the two liquids;

[0013] b. Centrifuge the centrifuge tube with the upper and lower layer liquids at a centrifugal force of 400 - 600 g for a predetermined time;

[0014] c. Discard the supernatant, add shark blood diluent to the centrifuge tube, mix evenly, and then centrifuge for a predetermined time;

[0015] d. Discard the supernatant, resuspend the cell mass at the bottom of the tube in shark blood diluent, centrifuge for a predetermined time, and repeat this step more than twice.

[0016] Preferably, the component of the shark blood diluent in each step is that 1 L of water contains 19.86 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, and 27.68 g of Urea, and the pH of the shark blood diluent is 7.4.

[0017] Preferably, after centrifugation in step b, four layers will appear from top to bottom in the separation tube. The first layer is the plasma layer, the second layer is the milky white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Use a disposable plastic Pasteur pipette to suck the milky white PBMC layer along the peripheral wall of the tube into a 15 mL centrifuge tube, and turn off the brake of the centrifuge each time during centrifugation to avoid destroying the density gradient during the deceleration process.

[0018] Preferably, in step b, the centrifuge tube is centrifuged at a centrifugal force of 500 g for about 30 minutes.

[0019] Preferably, in steps c and d, the centrifuge tube is centrifuged at a centrifugal force of 300 g for about 10 minutes.

[0020] Preferably, in step a, the fresh anticoagulated whole shark blood and the shark blood diluent are mixed in equal volume, and the mixed liquid is then added to the centrifuge tube in a volume ratio of 1:1 with the separation liquid.

[0021] Preferably, after completing step d, further discard the supernatant after centrifugation, and resuspend the cells in 0.1 - 0.2 mL of shark blood diluent for standby or process according to the requirements of the next experiment.

[0022] In the above-mentioned separating liquid and separation method for separating shark PBMC, the inventor has found through experiments that 100 mL of Ficoll-Urografin layering liquid contains 1-2 g of sodium chloride, more preferably 1.25-1.75 g, so that the density of the separating liquid is 1.090 g / mL - 1.095 g / mL, forming a separating liquid formula with an appropriate density. Among them, the Ficoll-Urografin layering liquid added with NaCl forms a density medium in the centrifuge tube. The shark blood cell suspension is placed on the top of the medium. Due to the difference in sedimentation coefficients between different cells, under the action of a certain centrifugal force, different cells settle at a certain speed respectively and form zones in different regions of the density gradient, so that different cells are layered and separated, and high-purity shark PBMC can be obtained. The components of this separating liquid are simple, the separation operation is simple and easy to carry out, and each component is a very stable substance. The prepared separating liquid is easy to store, simplifying the preparation process and storage conditions. Description of the Drawings

[0023] Figure 1 It is the separation effect diagram of shark PBMC directly using Ficoll-Urografin layering liquid (1.077 g / mL).

[0024] Figure 2 (a)-(c) are the separation effects of PBMC under different separating liquid compositions and centrifugal forces. Detailed Embodiments

[0025] The following will describe the present invention in detail with reference to specific embodiments and the drawings.

[0026] An embodiment of the present invention provides a separating liquid for separating shark PBMC. The separating liquid contains 1-2 g of NaCl in 100 mL of Ficoll-Urografin layering liquid, and the density of the separating liquid is 1.087 g / mL - 1.097 g / mL.

[0027] Preferably, the density of the layering liquid is 1.077 g / mL. The existing densities of Ficoll-Urografin layering liquid are 1.073 g / mL, 1.077 g / mL, and 1.084 g / mL. After continuous experiments, it is found that the density of 1.073 g / mL is too low and the density of 1.084 g / mL is too high, both of which are not suitable for density adjustment by an appropriate amount of NaCl.

[0028] Preferably, the separation liquid contains 1.25 - 1.75 g of NaCl in 100 mL of Ficoll - Hypaque stratification liquid. Correspondingly, the density of the separation liquid is 1.090 g / mL - 1.095 g / mL. Sodium chloride is an inorganic ionic compound with the chemical formula NaCl, which is easily soluble in solvents such as water and glycerol, has relatively good stability, its aqueous solution is neutral, and the density of the solution is greater than that of water. By adding NaCl, the density, osmotic pressure, etc. of the liquid can be precisely changed. In the embodiments of the present invention, the principle of separating shark PBMC is based on density gradient centrifugation, that is, a density medium is formed in the centrifuge tube by using Ficoll - Hypaque stratification liquid added with NaCl. The shark blood cell suspension is placed on the top of the medium. Due to the difference in sedimentation coefficients between different cells, under a certain centrifugal force, different cells sediment at a certain speed respectively and form zones in different regions of the density gradient, so that different cells are stratified and separated.

[0029] On the other hand, an embodiment of the present invention provides a separation method for separating shark PBMC, which includes the following steps:

[0030] a. After mixing fresh anticoagulated whole shark blood with shark blood diluent, add them to the centrifuge tube in a predetermined volume ratio with the separation liquid for separating shark PBMC as described above. First add the separation liquid, and then add the diluted shark blood, so that the two liquids are fixedly separated into upper and lower layers without mixing. The upper layer is the diluted shark blood, and the lower layer is the separation liquid, maintaining a clear interface between the two liquids.

[0031] b. Centrifuge the centrifuge tube with the upper and lower layer liquids at a centrifugal force of 400 - 600 g for a predetermined time;

[0032] c. Discard the supernatant, add shark blood diluent to the centrifuge tube, mix evenly, and then centrifuge for a predetermined time;

[0033] d. Discard the supernatant, resuspend the cell mass at the bottom of the tube in shark blood diluent, centrifuge for a predetermined time, and repeat this step more than twice.

[0034] In step a, the process of obtaining fresh anticoagulated whole shark blood is as follows: After disinfecting the caudal vein of the anesthetized shark with alcohol, use a 10 mL syringe pre-added with EDTA-Na2 anticoagulant to draw the required volume of shark venous blood, gently mix it, remove the syringe needle, and gently inject the venous blood sample into a centrifuge tube. To obtain good PBMC separation effect, the following operations are preferably completed within 2 hours and at room temperature. Preferably, the composition of the shark blood diluent in each step is 19.86 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, and 27.68 g of Urea in 1 L of water, and the pH of the shark blood diluent is 7.4. Preferably, in step a, the fresh anticoagulated whole shark blood and the shark blood diluent are mixed in equal volumes, and the mixed solution is then added to the centrifuge tube in a 1:1 volume ratio with the separation liquid.

[0035] The specific process of adding liquid to the centrifuge tube in step a is as follows: Dispense an appropriate volume of shark blood PBMC separation liquid into the centrifuge tube, and then carefully and slowly add an equal volume of diluted shark blood to the upper layer of the separation liquid, taking care not to mix the two liquids and maintaining a clear interface between the two liquids. If the volume of the diluted blood is less than 5 mL, use 5 mL of the separation liquid; if it is greater than 5 mL, use an equal volume of the separation liquid. To achieve good separation performance, the total volume of the two liquids should not exceed 2 / 3 of the centrifuge tube capacity.

[0036] Preferably, in step b, the centrifuge tube is preferably centrifuged at a centrifugal force of 500 g for about 30 minutes. After centrifugation in step b, four layers will appear from top to bottom in the separation tube. The first layer is the plasma layer, the second layer is the milky white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Use a disposable plastic Pasteur pipette to suck the milky white PBMC layer along the tube wall periphery and place it in a 15 mL centrifuge tube. Additionally, preferably, the brake of the centrifuge is turned off during each centrifugation to avoid damaging the density gradient during the deceleration process.

[0037] Preferably, in steps c and d, the centrifuge tube is preferably centrifuged at a centrifugal force of 300 g for about 10 minutes.

[0038] Preferably, after completing step d, further discard the supernatant after centrifugation, and resuspend the cells in 0.1 - 0.2 mL of shark blood diluent for standby or process according to the requirements of the next experiment.

[0039] In addition, in the embodiments of the present invention, according to the characteristics of shark blood, the spotted bamboo shark is used as the experimental object to explore the density of the shark PBMC separation liquid and the separation method.

[0040] The separation solution is prepared by adding an appropriate amount of NaCl to the polysucrose-diatrizoate layering solution, increasing the density of the separation solution from 1.077 g / mL to 1.087-1.097 g / mL, which can effectively separate and obtain high-purity PBMCs, and makes the composition and preparation method of the separation solution simpler, simplifies the operation steps, and makes the separation solution easier to store.

[0041] The following uses a number of examples to illustrate the separation method for isolating shark PBMCs, as well as the properties of the separation solution prepared.

[0042] Example 1: Preliminary exploration of the separation of PBMCs from the peripheral blood of bamboo sharks at different separation fluid densities and centrifugal speeds

[0043] (1) Reagent preparation

[0044] Preparation of shark blood dilution solution: weigh 19.86 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 27.68 g Urea, dissolve and mix in ultrapure water, adjust to pH 7.4, make up to 1 L, filter at 0.22 um, and store at 4 degrees.

[0045] Preparation of Shark PBMC Isolation Solution: Add 1.5g (A1, B1), 2g (A2, B2), 2.5g (A3, B3), 3g (A4, B4), 3.5g (A5, B5) of sodium chloride to each 100mL of polysucrose-diatrizoate layering solution (density 1.077g / mL), mix thoroughly, filter at 0.22um, and store at room temperature. The brackets indicate that they are divided into two identical groups, for example, (A1, B1) is two samples A1 and B1 taken from the same ratio of separation solution.

[0046] (2) Blood collection

[0047] Place the bamboo shark in seawater containing anesthetics and observe the shark's condition. If the shark breathes and swims slowly, flips over to expose its abdomen and is unable to flip, and is taken out of the water without struggling, blood sampling can be performed.

[0048] After disinfecting the shark's caudal vein with alcohol, use a 10mL syringe pre-added with EDTA-Na2 anticoagulant to draw the required volume of shark venous blood (30~50mL of whole blood can be collected from an adult striped bamboo shark), mix gently, remove the syringe needle, gently inject the venous blood sample into the centrifuge tube and slowly mix.

[0049] To achieve good PBMC isolation results, the remaining steps need to be completed within 2 hours at room temperature.

[0050] (3) PBMC isolation

[0051] A. Add fresh anticoagulated whole blood to an equal volume of shark diluent and mix well.

[0052] B. Aliquot 5 mL of shark PBMC separation liquid A1 - A5 and B1 - B5 into centrifuge tubes respectively. Then carefully and slowly add 5 mL of diluted shark blood to the upper layer of the separation liquid, taking care not to mix the two mixtures and maintaining a clear interface between the two liquids.

[0053] C. Set the centrifugation speeds to 1000 g (for groups A1 - A5) and 600 g (for groups B1 - B5) respectively, centrifuge for 30 min, and turn off the brake to prevent disruption of the density gradient during deceleration.

[0054] D. Observe the centrifugation effect of blood cells in each group after centrifugation. Four layers will appear from top to bottom: the first layer is the plasma layer, the second layer is the milky - white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Use a disposable plastic Pasteur pipette to aspirate the milky - white PBMC layer along the peripheral wall of the tube into a 15 - mL centrifuge tube.

[0055] E. Add 10 mL of diluent to the centrifuge tube, mix well, and centrifuge at 300 g for 10 min.

[0056] F. Discard the supernatant, resuspend the cells in 5 mL of diluent, and centrifuge at 300 g for 10 min. Repeat this step 2 times.

[0057] G. Discard the supernatant, resuspend the cells in 0.1 - 0.2 mL of diluent for standby or process according to the requirements of the next experiment.

[0058] The experimental results show that for the Ficoll - Hypaque layering solution (with a density of 1.092 - 1.112 g / mL) added with 1.5 - 3.5 g of sodium chloride, in both the A1 - A5 and B1 - B5 experimental groups, the smaller the density of the layering solution, the better the separation effect. The buffy coat layer (i.e., the PBMC layer) of A1 - A2 is diffuse, the buffy coat layer of A3 and B3 is less and mixed with red blood cells, and the buffy coat layer and the red blood cell layer of A4 - A5 and B4 - B5 are adhered together and the buffy coat layer cannot be aspirated cleanly.

[0059] The experimental results also show that the centrifugation speed has a great influence on the PBMC separation effect. The separation effect of the buffy coat layer at a centrifugal force of 600 g is significantly better than that at 1000 g. For example, the buffy coat layer of B1 - B2 is clearly stratified with a clear interface, while the buffy coat layer of A1 - A2 is cloud - like and more diffuse, and the number of cells at the bottom of the tube after cell washing and centrifugation is less than that of the B1 - B2 group, indicating a higher PBMC yield in the B1 - B2 group.

[0060] Based on the above experimental results, we further narrow down the range for optimizing the sodium chloride addition amount and the centrifugation speed to obtain a better PBMC separation effect.

[0061] Example 2: Optimize the density of the separation liquid and the centrifugation speed to improve the separation effect of peripheral blood PBMC of Chiloscyllium plagiosum

[0062] (1) Reagent preparation

[0063] The preparation of the shark blood diluent is the same as in Example 1.

[0064] Preparation of shark PBMC separation liquid: Add 0.75 g (C1, D1, E1), 1 g (C2, D2, E2), 1.25 g (C3, D3, E3), 1.5 g (C4, D4, E4), 1.75 g (C5, D5, E5) of sodium chloride to each 100 mL of Ficoll-Hypaque stratification solution (1.077 g / mL) respectively. After mixing well, filter through a 0.22 μm filter and store at room temperature.

[0065] (2) Blood collection

[0066] The blood collection method is the same as in Example 1.

[0067] (3) PBMC separation

[0068] A. Take the fresh anticoagulated whole blood of Chiloscyllium plagiosum, add an equal volume of shark diluent and mix well.

[0069] B. Aliquot 5 mL of shark PBMC separation liquid C1-C5, D1-D5, E1-E5 into centrifuge tubes respectively, and then carefully and slowly add 5 mL of diluted shark blood to the upper layer of the separation liquid. Note that the two mixtures should not be mixed, and a clear interface between the two liquids should be maintained.

[0070] C. Set the centrifugation speed to 600 g (group C1-C5), 500 g (group D1-D5), 400 g (group E1-E5) respectively, centrifuge for 30 min, and turn off the brake to prevent the density gradient from being damaged during deceleration.

[0071] D. Observe the centrifugation effect of each group of blood cells after centrifugation. Four layers will appear from top to bottom: the first layer is the plasma layer, the second layer is the milky white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Use a disposable plastic Pasteur pipette to aspirate the milky white PBMC layer along the peripheral wall of the tube and place it in a 15 mL centrifuge tube.

[0072] E. Add 10 mL of diluent to the centrifuge tube, mix well, and centrifuge at 300 g for 10 min.

[0073] F. Discard the supernatant, resuspend the cells in 5 mL of diluent, and centrifuge at 300 g for 10 min. Repeat this step 2 times.

[0074] G. Discard the supernatant, and resuspend the cells in 0.1 - 0.2 mL of diluent for standby or process according to the requirements of the next experiment.

[0075] The experimental results show that in the Ficoll - Hypaque density gradient medium (with a density of 1.085 - 1.095 g / mL) added with 0.75 - 1.75 g of sodium chloride, in the experimental groups C1 - C5, D1 - D5, and E1 - E5, as the density of the density gradient medium increased, the number of isolated white blood cells increased. Among them, at a centrifugal force of 500 g, the PBMC separation effect of group D3 was the best, with obvious stratification, clear interface, and no contamination of red blood cells. Followed by group D4 and group D5. In groups C1 - C2, D1 - D2, and E1 - E2, the white buffy coat could not be separated due to the too - low density of the separation medium.

[0076] The experimental results also show that at a centrifugal force of 500 g, the separation effect of the white buffy coat is better than that at 600 g and 400 g, with obvious stratification, clear interface, and a larger number of white blood cells. As shown in the following table, it shows the separation of the white buffy coat under different compositions of the density gradient medium and centrifugal forces.

[0077]

[0078] Figure 1 It shows the separation effect of shark PBMC directly using Ficoll - Hypaque density gradient medium (1.077 g / mL). It can be seen from the figure that the PBMC layer adheres to the red blood cell layer and cannot be separated.

[0079] Figure 2 It is the separation effect of PBMC under different compositions of the separation medium and centrifugal forces. As mentioned above, it is preferred to add 1.25 - 1.75 g of NaCl. Therefore, Figure 2 It mainly shows the actual effect of separating shark PBMC using the separation media formed by adding 1.25 g (C3, D3, E3), 1.5 g (C4, D4, E4), and 1.75 g (C5, D5, E5) of sodium chloride into 100 mL of Ficoll - Hypaque density gradient medium (1.077 g / mL).

[0080] From Table 1 and Figure 2 It can be concluded that after adding an appropriate amount of NaCl, at a centrifugal force of 500 g, the PBMC separation effect of group D3 is the best, with obvious stratification, clear interface, and no contamination of red blood cells. Followed by group D4 and group D5. In groups C1 - C2, D1 - D2, and E1 - E2, the white buffy coat could not be separated due to the too - low density of the separation medium.

[0081] It should be noted that the present invention is not limited to the above - mentioned embodiments. According to the creative spirit of the present invention, those skilled in the art can also make other changes, and these changes made according to the creative spirit of the present invention should be included within the scope of protection required by the present invention.

Claims

1. A separation solution for separating shark PBMC, characterized in that: The separation solution is prepared by adding 1.5-1.75 g NaCl to 100 mL of polysucrose-diatrizoate layering solution, and the density of the separation solution is 1.092 g / mL-1.095 g / mL.

2. The separation solution for separating shark PBMC according to claim 1, characterized in that: The density of the layered liquid is 1.077 g / mL.

3. A method for isolating shark PBMC, characterized in that: The following steps are involved: a. After mixing the fresh anticoagulated whole blood of shark with the shark blood diluent, add the separation solution for separating shark PBMC according to any one of claims 1 to 2 into a centrifuge tube according to a predetermined volume ratio, first add the separation solution, then add the diluted shark blood, so that the two liquids are fixedly separated into upper and lower layers without mixing each other, the upper layer is the diluted shark blood, and the lower layer is the separation solution, and a clear interface between the two liquids is maintained; b. Centrifuge the centrifuge tube with two layers of liquid at 500-600g centrifugal force for a predetermined time; c. Discard the supernatant, add shark blood dilution to the centrifuge tube, mix well, and centrifuge for a predetermined time; d. Discard the supernatant, resuspend the cell pellet at the bottom of the tube in shark blood diluent, centrifuge for a predetermined time, and repeat this step twice or more.

4. The method for isolating shark PBMC according to claim 3, characterized in that: The components of the shark blood dilution in each step are 19.86 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 27.68 g Urea in 1 L of water, and the pH of the shark blood dilution is 7.

4.

5. The method for isolating shark PBMC according to claim 3, characterized in that: After centrifugation in step b, four layers will appear in the separation tube from top to bottom. The first layer is the plasma layer, the second layer is the milky white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Use a disposable plastic Pasteur tube to draw the milky white PBMC layer along the edge of the tube wall and place it in a 15 mL centrifuge tube. Turn off the brake of the centrifuge each time centrifuging to avoid destroying the density gradient during the deceleration process.

6. The method for isolating shark PBMC according to claim 3, characterized in that: In step b, the centrifuge tube is centrifuged at 500 g.

7. The method for isolating shark PBMC according to claim 3, characterized in that: In step c and step d, the centrifuge tube is centrifuged at 300 g centrifugal force.

8. The method for isolating shark PBMC according to claim 3, characterized in that: In step a, the shark fresh anticoagulated whole blood and the shark blood diluent are mixed in equal volumes, and the mixed solution is then mixed with the separation solution in a volume ratio of 1:1 and added into a centrifuge tube.

9. The method for isolating shark PBMC according to claim 3, characterized in that: After completing step d, the supernatant after centrifugation is further discarded, and the cells are resuspended in 0.1-0.2 mL of shark blood diluent for later use or processed according to the requirements of the next experiment.

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