Method for efficiently capturing neutrophils in peripheral blood by single-cell sequencing with erythrocyte lysis

Abstract

The present invention relates to the field of biomedical technologies and provides a method for efficiently capturing neutrophils from peripheral blood for single-cell sequencing by lysing red blood cells. The method includes the following steps: collecting human peripheral blood into an anticoagulant tube containing ethylenediaminetetraacetic acid; taking the ethylenediaminetetraacetic acid anticoagulated whole blood, adding a red blood cell lysate, lysing at room temperature, waiting until the turbid mixed blood solution becomes clear and transparent, waiting for 1 minute, then adding a FACS cell resuspension solution to terminate red blood cell lysis, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding a phosphate buffer solution to slowly resuspend the cells, and centrifuging. By selecting a suitable anticoagulant, precisely controlling the red blood cell lysis conditions, and performing meticulous cell resuspension and centrifugation operations, the present invention can ensure high neutrophil activity and a low cell debris rate, provide a suitable single-cell suspension for single-cell RNA sequencing, and help analyze the characteristics and functions of various neutrophil subsets.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for lysing red blood cells in peripheral blood for highly efficient capture of neutrophils by single-cell sequencing. Background Art

[0002] Neutrophils are a type of myeloid leukocytes with a series of important functions and are important effector cells in the innate immune response. Under normal physiological conditions in healthy individuals, neutrophils account for 50%-70% of circulating leukocytes. However, during infection or acute injury, the production rate of neutrophils increases significantly to participate in the relevant immune processes. It can be seen that neutrophils play an important role in human health and disease regulation. Among human circulating leukocytes, the subsets and differentiation lineages of neutrophils have not been fully characterized, and the proportions and functions of cells in each subset are still not fully clear.

[0003] Single-cell RNA sequencing (scRNA-seq) can deeply reveal the characteristics of different subpopulations of cells in human diseases, provide research on the pathogenesis of diseases, and have potential clinical guiding value. The basic steps of single-cell RNA sequencing include the preparation of single-cell suspensions, the capture of RNA molecules, reverse transcription, cDNA amplification, library preparation, sequencing, and data analysis. As is well known, the preparation of single-cell suspensions is the key first step in single-cell sequencing, in which the cell viability of the cell suspension should reach more than 90%, and cell clumps or debris should be less than 10%. Single-cell RNA sequencing can be used to analyze various immune cells in whole blood, including the proportions and functional characteristics of each subset of neutrophils, so as to reveal the pathogenic mechanisms of different subsets of neutrophils in regulating diseases and provide 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 is no report on the method for lysing red blood cells in peripheral blood for capturing neutrophils by single-cell sequencing. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a method for lysing red blood cells in peripheral blood for highly efficient capture of neutrophils by single-cell sequencing, aiming to solve the problems mentioned in the background art.

[0005] The present invention provides a method for lysing red blood cells in peripheral blood for highly efficient capture of neutrophils by single-cell sequencing, including the following steps:

[0006] Step S1: Collect 0.5-1 mL of human peripheral blood into an anticoagulation tube containing ethylenediaminetetraacetic acid, gently shake and mix to prevent blood coagulation, and obtain ethylenediaminetetraacetic acid anticoagulated whole blood;

[0007] Step S2: Take ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood into a centrifuge tube, add 6 - 7 times the volume of red blood cell lysate, gently shake and mix well, then lyse at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent. After waiting for 1 minute, add 3 - 4 mL of FACS cell resuspension solution to terminate red blood cell lysis, and centrifuge.

[0008] Step S3: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge.

[0009] Step S4: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge.

[0010] Step S5: Discard the supernatant, add 4 - 5 mL of phosphate buffer to slowly resuspend the cells, and centrifuge.

[0011] Further, in step S1, the amount of ethylenediaminetetraacetic acid is 1.0 - 2.2 mg.

[0012] Further, in step S2, take 0.5 mL of ethylenediaminetetraacetic acid anticoagulated whole blood.

[0013] Further, in step S2, the centrifugation conditions are 300 g, 3 minutes, and 10 °C.

[0014] Further, in step S3, the centrifugation conditions are 250 g, 3 minutes, and 10 °C.

[0015] Further, in step S4, the centrifugation conditions are 200 g, 3 minutes, and 10 °C.

[0016] Further, in step S5, the centrifugation conditions are 200 g, 3 minutes, and 10 °C.

[0017] Further, during the centrifugation process from step S2 to step S5, the angular acceleration of the centrifuge during acceleration and deceleration ≤ 5.3 rad / s 2 。

[0018] The present invention has the following beneficial effects:

[0019] (1) By selecting a suitable anticoagulant, precisely controlling the red blood cell lysis conditions, and performing careful cell resuspension and centrifugation operations, it is possible to ensure high neutrophil activity and a low cell debris rate, providing a suitable single - cell suspension for single - cell RNA sequencing, which helps to analyze the characteristics and functions of various neutrophil subpopulations.

[0020] (2) Add red blood cell lysate at a volume 6 - 7 times that of the blood sample. If the ratio is too low, the purpose of lysing red blood cells cannot be achieved; if the ratio is too high, cells other than red blood cells in peripheral blood (especially neutrophils) are likely to be damaged. Wait until the turbid mixed blood solution becomes clear and transparent, and then start the lysis process after waiting for 1 minute. Precise control of the termination time of red blood cell lysis can not only achieve the purpose of red blood cell lysis but also ensure high activity and low fragmentation rate of immune cells including neutrophils. Strictly control the centrifugation conditions and the angular acceleration of the centrifuge during speed increase and decrease to prevent damage to cells (especially neutrophils) caused by too fast speed increase and decrease. Brief Description of the Drawings

[0021] The exemplary embodiments of the present invention can be more fully understood by referring to the following drawings:

[0022] 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 blood cell lysis process. Detailed Embodiments

[0023] In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the 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.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0025] The embodiments of the present invention provide a method for lysing red blood cells in peripheral blood for efficient capture of neutrophils in single-cell sequencing, including the following steps:

[0026] Step S1: Collect 0.5 - 1 mL of human peripheral blood into an anticoagulation tube containing ethylenediaminetetraacetic acid (EDTA), gently shake and mix to prevent blood coagulation, and obtain EDTA-anticoagulated whole blood;

[0027] Step S2: Transfer the EDTA-anticoagulated whole blood into a centrifuge tube, add red blood cell lysate at a volume 6 - 7 times that of the blood sample, gently shake and mix, and then perform lysis at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent, and after waiting for 1 minute, add 3 - 4 mL of FACS cell resuspension solution to terminate the red blood cell lysis, and then centrifuge;

[0028] Step S3: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and then centrifuge;

[0029] Step S4: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and then centrifuge;

[0030] Step S5: Discard the supernatant, add 4 - 5 mL of phosphate buffer to resuspend the cells slowly, and centrifuge.

[0031] In some embodiments, in step S1, the ethylenediaminetetraacetic acid is 1.0 - 2.2 mg.

[0032] In some embodiments, in step S2, take 0.5 mL of ethylenediaminetetraacetic acid anticoagulated whole blood.

[0033] In some embodiments, in step S2, the centrifugation conditions are 300 g, 3 minutes, and 10 °C.

[0034] In some embodiments, in step S3, the centrifugation conditions are 250 g, 3 minutes, and 10 °C.

[0035] In some embodiments, in step S4, the centrifugation conditions are 200 g, 3 minutes, and 10 °C.

[0036] In some embodiments, in step S5, the centrifugation conditions are 200 g, 3 minutes, and 10 °C.

[0037] In some embodiments, during the centrifugation process from step S2 to step S5, the angular acceleration of the centrifuge during speed increase and decrease ≤ 5.3 rad / s 2 , to prevent damage to cells caused by too fast speed increase and decrease.

[0038] Among them, when resuspending cells in steps S3 - S5, all operations are performed on ice and gently to avoid damaging cells.

[0039] Experimental reagents:

[0040] (1) FACS cell resuspension solution: Prepared from 19.6 mL of 1 - fold concentration phosphate buffer (product number: SH30256.01) and 400 μl fetal bovine serum (product number: A3160802), and used immediately after preparation;

[0041] (2) Red blood cell lysis solution: Prepared from 8.29 g of ammonium chloride (product number: A801304), 1 g of potassium bicarbonate (product number: P816192), 0.0372 g of ethylenediaminetetraacetic acid (E809068) and 1 L of distilled water.

[0042] Example 1:

[0043] (1) Collect 1 mL of human peripheral blood into an anticoagulant tube containing 2.2 mg of ethylenediaminetetraacetic acid, gently shake and mix to prevent blood coagulation, and obtain ethylenediaminetetraacetic acid anticoagulated whole blood;

[0044] (2) Take 0.5 mL of ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood into a centrifuge tube, add 3.5 mL of red blood cell lysate, gently shake and mix well, then lyse at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent. After waiting for 1 minute, add 3 mL of FACS cell resuspension solution to terminate red blood cell lysis. Centrifuge at a condition of 300 g for 3 minutes at 10 °C. Resuspend the cells on ice with gentle operation, and the angular acceleration of the centrifuge during speed up and slow down ≤ 5.3 rad / s 2 ;

[0045] (3) Discard the supernatant, add 4 mL of FACS cell resuspension solution to slowly resuspend the cells, centrifuge at a condition of 250 g for 3 minutes at 10 °C. Resuspend the cells on ice with gentle operation, and the angular acceleration of the centrifuge during speed up and slow down ≤ 5.3 rad / s 2 ;

[0046] (4) Discard the supernatant, add 4 mL of FACS cell resuspension solution to slowly resuspend the cells, centrifuge at a condition of 200 g for 3 minutes at 10 °C. Resuspend the cells on ice with gentle operation, and the angular acceleration of the centrifuge during speed up and slow down ≤ 5.3 rad / s 2 ;

[0047] (5) Discard the supernatant, add 4 mL of phosphate buffer solution to slowly resuspend the cells, centrifuge at a condition of 200 g for 3 minutes at 10 °C. Resuspend the cells on ice with gentle operation, and the angular acceleration of the centrifuge during speed up and slow down ≤ 5.3 rad / s 2 。

[0048] The state diagram when the turbid mixed blood solution becomes clear and transparent during red blood cell lysis is as Figure 1 shown.

[0049] Cell viability and cell fragmentation were evaluated under a microscope. The results showed that the cell viability was 99.96% and the cell fragmentation rate was 6.10%. After library construction, single-cell RNA sequencing was performed, and the sequencing results showed that the captured neutrophils accounted for 64%.

[0050] Example 2:

[0051] (1) Collect 0.5 mL of human peripheral blood into an anticoagulation tube containing 1.0 mg of ethylenediaminetetraacetic acid (EDTA), gently shake and mix well to prevent blood coagulation, and obtain EDTA-anticoagulated whole blood;

[0052] (2) Take 0.5 mL of EDTA-anticoagulated whole blood into a centrifuge tube, add 3.0 mL of red blood cell lysate, gently shake and mix well, then lyse at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent. After waiting for 1 minute, add 4 mL of FACS cell resuspension solution to terminate red blood cell lysis. Centrifuge at a condition of 300 g for 3 minutes at 10 °C. Resuspend the cells on ice with gentle operation, and the angular acceleration of the centrifuge during speed up and slow down ≤ 5.3 rad / s2 ;

[0053] (3) Discard the supernatant, add 4 mL of FACS cell resuspension solution, and gently resuspend the cells. Then centrifuge the cells under the conditions of 250 g, 3 minutes, and 10 °C. Resuspend the cells on ice, with gentle operation. The angular acceleration of the centrifuge during speed-up and slow-down should be ≤ 5.3 rad / s 2 ;

[0054] (4) Discard the supernatant, add 5 mL of FACS cell resuspension solution, and gently resuspend the cells. Then centrifuge the cells under the conditions of 200 g, 3 minutes, and 10 °C. Resuspend the cells on ice, with gentle operation. The angular acceleration of the centrifuge during speed-up and slow-down should be ≤ 5.3 rad / s 2 ;

[0055] (5) Discard the supernatant, add 5 mL of phosphate buffer solution, and gently resuspend the cells. Then centrifuge the cells under the conditions of 200 g, 3 minutes, and 10 °C. Resuspend the cells on ice, with gentle operation. The angular acceleration of the centrifuge during speed-up and slow-down should be ≤ 5.3 rad / s 2 .

[0056] Evaluate cell viability and cell fragmentation under a microscope. The results show that the cell viability is 97.00% and the cell fragmentation rate is 4.00%. After library construction, single-cell RNA sequencing is performed, and the sequencing results show that neutrophils captured account for 40%.

[0057] Example 3:

[0058] (1) Collect 0.8 mL of human peripheral blood into an anticoagulation tube containing 1.7 mg of ethylenediaminetetraacetic acid, gently shake and mix to prevent blood coagulation, and obtain ethylenediaminetetraacetic acid anticoagulated whole blood;

[0059] (2) Take 0.5 mL of ethylenediaminetetraacetic acid anticoagulated whole blood into a centrifuge tube, add 3.5 mL of red blood cell lysis solution, gently shake and mix, and then lyse at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent. After waiting for 1 minute, add 3 mL of FACS cell resuspension solution to terminate red blood cell lysis. Then centrifuge the cells under the conditions of 300 g, 3 minutes, and 10 °C. Resuspend the cells on ice, with gentle operation. The angular acceleration of the centrifuge during speed-up and slow-down should be ≤ 5.3 rad / s 2 ;

[0060] (3) Discard the supernatant, add 5 mL of FACS cell resuspension solution, and gently resuspend the cells. Then centrifuge the cells under the conditions of 250 g, 3 minutes, and 10 °C. Resuspend the cells on ice, with gentle operation. The angular acceleration of the centrifuge during speed-up and slow-down should be ≤ 5.3 rad / s 2 ;

[0061] (4) Discard the supernatant, add 4 mL of FACS cell resuspension solution, and slowly resuspend the cells. Centrifuge the cells at 200 g for 3 minutes at 10 °C. Resuspend the cells on ice gently. The angular acceleration of the centrifuge during acceleration and deceleration should be ≤ 5.3 rad / s. 2 ;

[0062] (5) Discard the supernatant, add 5 mL of phosphate buffer solution, and slowly resuspend the cells. Centrifuge the cells at 200 g for 3 minutes at 10 °C. Resuspend the cells on ice gently. The angular acceleration of the centrifuge during acceleration and deceleration should be ≤ 5.3 rad / s. 2 。

[0063] Evaluate cell viability and cell fragmentation under a microscope. The results show that the cell viability is 99.60% and the cell fragmentation rate is 6.60%. After library construction, single-cell RNA sequencing is performed. The sequencing results show that neutrophils captured account for 39%.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for lysing red blood cells in peripheral blood to efficiently capture neutrophils by single-cell sequencing, characterized in that: It includes the following steps: Step S1: Collect 0.5 - 1 mL of human peripheral blood into an anticoagulation tube containing ethylenediaminetetraacetic acid (EDTA), gently shake and mix well to obtain EDTA anticoagulated whole blood. Step S2: Take the EDTA anticoagulated whole blood into a centrifuge tube, add 6 - 7 times the volume of red blood cell lysate, gently shake and mix well, then lyse at room temperature. Wait until the turbid mixed blood solution becomes clear and transparent. After waiting for 1 minute, add 3 - 4 mL of FACS cell resuspension solution to terminate red blood cell lysis, and centrifuge. The centrifugation conditions are 300 g, 3 minutes, and 10 °C. Step S3: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge. The centrifugation conditions are 250 g, 3 minutes, and 10 °C. Step S4: Discard the supernatant, add 4 - 5 mL of FACS cell resuspension solution to slowly resuspend the cells, and centrifuge. The centrifugation conditions are 200 g, 3 minutes, and 10 °C. Step S5: Discard the supernatant, add 4 - 5 mL of phosphate buffer solution to slowly resuspend the cells, and centrifuge. The centrifugation conditions are 200 g, 3 minutes, and 10 °C. Among them, the FACS cell resuspension solution is prepared from 19.6 mL of 1-fold concentration phosphate buffer solution and 400 μL of fetal bovine serum, and it should be used immediately after preparation. During the centrifugation process from Step S2 to Step S5, the angular acceleration of the centrifuge during acceleration and deceleration ≤ 5.3 rad / s 2 .

2. The method for lysing red blood cells in peripheral blood to efficiently capture neutrophils 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 neutrophils from peripheral blood for single-cell sequencing according to claim 1, characterized in that: In step S2, take 0.5 mL of EDTA anticoagulated whole blood.

Images