Peripheral blood crack red method for efficiently capturing neutrophil through single cell sequencing

Abstract

The invention relates to the technical field of biological medicine, and provides a peripheral blood crack red method for efficiently capturing neutrophil through single cell sequencing. Comprising the following steps: collecting human peripheral blood into an ethylenediaminetetraacetic acid-containing anticoagulant tube; the method comprises the following steps: taking ethylenediamine tetraacetic acid anticoagulant whole blood, adding a red blood cell lysis buffer, carrying out lysis at room temperature, waiting for 1 minute after a turbid mixed blood solution becomes clear and transparent, adding an FACS cell resuspension to terminate lysis red, and carrying out centrifugation; adding an FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding an FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; and adding a phosphate buffer solution to slowly resuspend the cells, and centrifuging. According to the method disclosed by the invention, the high activity and low cell debris rate of the neutrophile granulocyte can be ensured by selecting the proper anticoagulant, accurately controlling the erythrocyte lysis condition and careful cell resuspension and centrifugal operation, a proper single-cell suspension is provided for single-cell RNA sequencing, and the analysis of the characteristics and functions of various subgroups of the neutrophile granulocyte is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a peripheral blood lysis method for efficiently capturing neutrophils by single-cell sequencing. Background Art

[0002] Neutrophils are a type of myeloid leukocytes with a series of important functions, and they serve as important effector cells of the innate immune response. Under normal physiological conditions in healthy people, neutrophils account for 50%-70% of circulating leukocytes. However, in cases of infection or acute injury, the rate of neutrophil production increases significantly and participates in related immune processes. It can be seen that neutrophils play an important role in regulating human health and disease. Among human circulating leukocytes, the subpopulations of neutrophils and their differentiation lineages have not been fully characterized, and the proportion and function of each subpopulation of cells are still not fully clear.

[0003] Single-cell RNA sequencing (scRNA-seq) can deeply reveal the characteristics of different cell subpopulations in human diseases, provide research on the pathogenesis of diseases, and has potential clinical guidance value. The basic steps of single-cell RNA sequencing include preparation of single-cell suspension, capture of RNA molecules, reverse transcription, cDNA amplification, library preparation, sequencing, and data analysis. It is well known that the preparation of single-cell suspension is the key first step in single-cell sequencing, in which the cell viability of the cell suspension should reach more than 90%, and the cell clumps or fragments should be less than 10%. Single-cell RNA sequencing can be used to analyze various immune cells in whole blood, including the proportion and functional characteristics of each subpopulation of neutrophils, thereby revealing the pathogenic mechanism of different subpopulations of neutrophils in regulating diseases and providing scientific guidance for the clinical diagnosis and treatment of diseases. However, neutrophils are very fragile, and it is extremely challenging to prepare single-cell suspensions containing neutrophils. At present, there are no reports on the peripheral blood lysis method for capturing neutrophils for single-cell sequencing. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for efficiently capturing peripheral blood lysis of neutrophils by single-cell sequencing, which aims to solve the problems mentioned in the background technology.

[0005] The present invention provides a method for efficiently capturing neutrophil peripheral blood lysis by single cell sequencing, comprising the following steps: Step S1: Collect 0.5-1 mL of human peripheral blood into an EDTA anticoagulant tube, shake gently to mix, and prevent blood coagulation to obtain EDTA anticoagulant whole blood; Step S2: Take EDTA anticoagulated whole blood into a centrifuge tube, add 6-7 times the volume of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. After the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 3-4 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge; Step S3: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S4: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S5: Discard the supernatant, add 4-5 mL of phosphate buffer to slowly resuspend the cells, and centrifuge.

[0006] Furthermore, in step S1, the amount of ethylenediaminetetraacetic acid is 1.0-2.2 mg.

[0007] Furthermore, in step S2, 0.5 mL of ethylenediaminetetraacetic acid anticoagulated whole blood is taken.

[0008] Furthermore, in step S2, the centrifugation conditions are 300 g, 3 minutes, and 10°C.

[0009] Furthermore, in step S3, the centrifugation conditions are 250 g, 3 minutes, and 10°C.

[0010] Furthermore, in step S4, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

[0011] Furthermore, in step S5, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

[0012] Furthermore, during the centrifugation process from step S2 to step S5, the angular acceleration of the centrifuge is ≤5.3 rad / s 2 .

[0013] The present invention has the following beneficial effects: (1) By selecting appropriate anticoagulants, precisely controlling red blood cell lysis conditions, and performing meticulous cell resuspension and centrifugation operations, high neutrophil activity and low cell fragmentation rates can be ensured, providing a suitable single-cell suspension for single-cell RNA sequencing, which helps to analyze the characteristics and functions of various neutrophil subpopulations.

[0014] (2) Add 6-7 times the volume of red blood cell lysis buffer. If the ratio is too low, the purpose of red blood cell lysis cannot be achieved, and if the ratio is too high, it is easy to damage cells other than red blood cells in peripheral blood (especially neutrophils). Wait for 1 minute after the turbid mixed blood solution becomes clear and transparent before red blood cell lysis. Accurately grasp the timing of red blood cell lysis to achieve the purpose of red blood cell lysis and ensure that the activity of immune cells including neutrophils is high and the fragmentation rate is low. Strictly control the centrifugal conditions and limit the angular acceleration of the centrifuge to prevent damage to cells (especially neutrophils) caused by excessive acceleration and deceleration. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This is a state diagram of Example 1 of the present invention when the turbid mixed blood solution becomes clear and transparent during the red splitting process. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0018] The embodiment of the present invention provides a method for efficiently capturing peripheral blood lysis of neutrophils by single-cell sequencing, comprising the following steps: Step S1: Collect 0.5-1 mL of human peripheral blood into an EDTA anticoagulant tube, shake gently to mix, and prevent blood coagulation to obtain EDTA anticoagulant whole blood; Step S2: Take EDTA anticoagulated whole blood into a centrifuge tube, add 6-7 times the volume of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. After the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 3-4 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge; Step S3: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S4: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S5: Discard the supernatant, add 4-5 mL of phosphate buffer to slowly resuspend the cells, and centrifuge.

[0019] In some embodiments, in step S1, the amount of EDTA is 1.0-2.2 mg.

[0020] In some embodiments, in step S2, 0.5 mL of EDTA-anticoagulated whole blood is collected.

[0021] In some embodiments, in step S2, the centrifugation conditions are 300 g, 3 minutes, and 10°C.

[0022] In some embodiments, in step S3, the centrifugation conditions are 250 g, 3 minutes, and 10°C.

[0023] In some embodiments, in step S4, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

[0024] In some embodiments, in step S5, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

[0025] In some embodiments, during the centrifugation process of step S2 to step S5, the angular acceleration of the centrifuge is ≤5.3 rad / s 2 , to prevent damage to cells caused by excessive speed increase or decrease.

[0026] The resuspending of cells in steps S3 to S5 is all performed on ice and the movements are gentle to avoid damaging the cells.

[0027] Experimental reagents: (1) FACS cell resuspension solution: Prepared from 19.6 mL of 1x phosphate buffer (Cat. No. SH30256.01) and 400 μl of fetal bovine serum (Cat. No. A3160802), ready for use; (2) Red blood cell lysis solution: Prepared with 8.29 g ammonium chloride (Cat. No. A801304), 1 g potassium bicarbonate (Cat. No. P816192), 0.0372 g ethylenediaminetetraacetic acid (E809068) and 1 L distilled water.

[0028] Embodiment 1: (1) Collect 1 mL of human peripheral blood into an anticoagulant tube containing 2.2 mg of EDTA, shake gently to mix, and prevent blood coagulation to obtain EDTA anticoagulated whole blood; (2) Take 0.5 mL of EDTA anticoagulated whole blood into a centrifuge tube, add 3.5 mL of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. When the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 3 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge at 300g, 3 minutes, 10°C. Resuspend the cells on ice and move gently. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (3) Discard the supernatant, add 4 mL of FACS cell resuspension buffer, slowly resuspend the cells, and centrifuge at 250 g for 3 minutes at 10°C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (4) Discard the supernatant, add 4 mL of FACS cell resuspension buffer and slowly resuspend the cells. Centrifuge at 200 g, 3 min, 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (5) Discard the supernatant, add 4 mL of phosphate buffer and slowly resuspend the cells. Centrifuge at 200 g for 3 minutes at 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 .

[0029] The state of the turbid mixed blood solution when it turns clear and transparent during the red splitting process is shown in the figure Figure 1 shown.

[0030] Cell activity and cell fragmentation were evaluated under a microscope, and the results showed that the cell activity was 99.96% and the cell fragmentation rate was 6.10%. Single-cell RNA sequencing was performed after library construction, and the sequencing results showed that the captured neutrophils accounted for 64%.

[0031] Embodiment 2: (1) Collect 0.5 mL of human peripheral blood into an anticoagulant tube containing 1.0 mg of EDTA, shake gently to mix, and prevent blood coagulation to obtain EDTA anticoagulated whole blood; (2) Take 0.5 mL of EDTA anticoagulated whole blood into a centrifuge tube, add 3.0 mL of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. When the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 4 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge at 300g, 3 minutes, 10°C. Resuspend the cells on ice and move gently. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (3) Discard the supernatant, add 4 mL of FACS cell resuspension buffer, slowly resuspend the cells, and centrifuge at 250 g for 3 minutes at 10°C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (4) Discard the supernatant, add 5 mL of FACS cell resuspension buffer and slowly resuspend the cells. Centrifuge at 200 g, 3 min, 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (5) Discard the supernatant, add 5 mL of phosphate buffer and slowly resuspend the cells. Centrifuge at 200 g for 3 minutes at 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 .

[0032] Cell activity and cell fragmentation were evaluated under a microscope, and the results showed that the cell activity was 97.00% and the cell fragmentation rate was 4.00%. Single-cell RNA sequencing was performed after library construction, and the sequencing results showed that the captured neutrophils accounted for 40%.

[0033] Embodiment 3: (1) Collect 0.8 mL of human peripheral blood into an anticoagulant tube containing 1.7 mg of EDTA, shake gently to mix, and prevent blood coagulation to obtain EDTA anticoagulant whole blood; (2) Take 0.5 mL of EDTA anticoagulated whole blood into a centrifuge tube, add 3.5 mL of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. When the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 3 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge at 300g, 3 minutes, 10°C. Resuspend the cells on ice and move gently. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (3) Discard the supernatant, add 5 mL of FACS cell resuspension buffer, slowly resuspend the cells, and centrifuge at 250 g for 3 minutes at 10°C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (4) Discard the supernatant, add 4 mL of FACS cell resuspension buffer and slowly resuspend the cells. Centrifuge at 200 g, 3 min, 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 ; (5) Discard the supernatant, add 5 mL of phosphate buffer and slowly resuspend the cells. Centrifuge at 200 g for 3 minutes at 10 °C. Resuspend the cells on ice and be gentle. The angular acceleration of the centrifuge should be ≤5.3 rad / s. 2 .

[0034] Cell activity and cell fragmentation were evaluated under a microscope, and the results showed that the cell activity was 99.60% and the cell fragmentation rate was 6.60%. Single-cell RNA sequencing was performed after library construction, and the sequencing results showed that the captured neutrophils accounted for 39%.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficiently capturing neutrophil lysate from peripheral blood by single-cell sequencing, characterized in that: The following steps are involved: Step S1: Collect 0.5-1 mL of human peripheral blood into an EDTA anticoagulant tube, and gently shake to mix to obtain EDTA anticoagulant whole blood; Step S2: Take EDTA anticoagulated whole blood into a centrifuge tube, add 6-7 times the volume of red blood cell lysis buffer, shake gently to mix, and lyse at room temperature. After the turbid mixed blood solution becomes clear and transparent, wait for 1 minute, add 3-4 mL of FACS cell resuspension buffer to stop red blood cell lysis, and centrifuge; Step S3: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S4: discard the supernatant, add 4-5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge; Step S5: Discard the supernatant, add 4-5 mL of phosphate buffer to slowly resuspend the cells, and centrifuge.

2. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S1, the amount of ethylenediaminetetraacetic acid is 1.0-2.2 mg.

3. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S2, 0.5 mL of EDTA-anticoagulated whole blood is taken.

4. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S2, the centrifugation conditions are 300 g, 3 minutes, and 10°C.

5. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S3, the centrifugation conditions are 250 g, 3 minutes, and 10°C.

6. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S4, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

7. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: In step S5, the centrifugation conditions are 200 g, 3 minutes, and 10°C.

8. The method for efficiently capturing neutrophil lysis by single-cell sequencing according to claim 1, characterized in that: During the centrifugation process from step S2 to step S5, the angular acceleration of the centrifuge is ≤5.3 rad / s 2 .

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