Establishment method for characteristics of pulmonary immune cell atlas in mice with snake venom-induced acute lung injury

Abstract

The present invention provides a method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury, comprising the following steps: S1 extracting immune cells; S2 coupling; S3 staining; S4 bioinformatics analysis; The beneficial effects of the present invention: The lung tissue is evenly minced and dissociated, centrifuged according to the cell density, and a large number of immune cells are separated. Washing is carried out using a high-glucose medium, which maximally ensures the yield and activity of pulmonary immune cells; The viability of the obtained cells can be known through trypan blue staining, and it is basically greater than 90%; 42 antibody detections can be simultaneously performed on mouse pulmonary immune cells, the cell-specific markers and functional proteins are detected, and the dynamic changes of pulmonary cell subsets can be observed.

Description

Technical Field

[0001] The present invention relates to the technical field of establishing immune cell characteristic maps, and particularly to a method for establishing the characteristic map of immune cells in the lungs of mice with acute lung injury caused by snake venom. Background Art

[0002] Snakebite is a neglected tropical disease that causes great pain, disability, and premature death to people in many countries and poor regions. According to the report of the WHO in 2019, about 4.5 million - 5.4 million people are bitten by snakes every year, among which 1.8 million - 2.7 million people are bitten by venomous snakes, resulting in 81,000 - 138,000 deaths and leaving 400,000 people with lifelong disabilities.

[0003] Daboia russelii is a highly venomous snake with an extremely fast onset of toxicity. The bitten patients will develop acute respiratory failure, and even progress to acute respiratory distress syndrome and multiple organ failure, endangering their lives. Snake venom enters the body, causing a large number of alveolar epithelial cells and capillary endothelial cells in the lungs to be damaged within a short time, inducing an inflammatory response. Currently, there is a lack of effective drugs for treating snakebite, and only rescue can be carried out based on clinical symptoms and treatment experience. Therefore, it is very important to explore the key regulatory molecular mechanism of Daboia russelii snake venom on acute lung injury.

[0004] When Daboia russelii snake venom induces acute lung injury, a large number of immune cells infiltrate the lungs, and these immune cells further aggravate the degree of lung injury by secreting a large number of chemokines and inflammatory factors. As time goes by, the lung injury worsens, and different immune cells play different roles at different stages. Exploring the changes of these immune cells is of great significance for the development of new drugs for treating snakebite. The mass cytometry technology used in this patent has 135 detection channels, can simultaneously label 42 antibodies for detection at one time, and has an extremely low background, and there is no interference between channels, overcoming the disadvantages of traditional flow cytometry. Through mass cytometry technology, we can quickly obtain the subset ratio and functional characteristics of immune cells in the lungs. Summary of the Invention

[0005] The present invention aims at the deficiencies of the prior art and provides a method for establishing the characteristic map of immune cells in the lungs of mice with acute lung injury caused by snake venom.

[0006] The present invention realizes the solution of the above technical problems through the following technical means:

[0007] A method for establishing the characteristic map of immune cells in the lungs of mice with acute lung injury caused by snake venom, comprising the following steps:

[0008] S1 Extract immune cells

[0009] Mice with acute lung injury caused by intraperitoneal injection of Daboia russelii venom were sacrificed at 6 h, perfused through the heart, and the lungs were removed after perfusing out the internal blood; the lungs were minced and mixed with dissociation solution, digested in a water bath for 30 min, and then subjected to density gradient centrifugation and red blood cell lysis to obtain pure mouse lung immune cells;

[0010] S2 Conjugation

[0011] Using an antibody conjugation kit, a metal isotope was linked to an antibody against a mouse lung immune cell marker to obtain a metal-labeled antibody;

[0012] S3 Staining

[0013] The isolated mouse lung immune cells were incubated with the metal-labeled antibody to label the immune cells;

[0014] S4 Bioinformatics Analysis

[0015] The labeled mouse lung immune cells were analyzed by mass cytometry. The t-SNE and phenograph algorithms were used to reduce the dimension and cluster the obtained data; then the expression distributions of multiple detection antibodies of different cell subsets were displayed on the same heat map, and the expression levels of different markers in different cell subsets and the proportion of cell subsets in the total cell number were shown through the t-SNE map, so as to represent the classification and functional observation of mouse lung immune cells.

[0016] As an improvement of the above technical solution, the S1 includes the following steps:

[0017] 1.1 Mice with acute lung injury caused by intraperitoneal injection of Daboia russelii venom were sacrificed after 6 h, wiped with 75% alcohol cotton, and the chest was cut open;

[0018] 1.2 Heart perfusion was performed, and normal saline was continuously perfused through the heart to remove the internal blood and turn the lung tissue from blood red to white;

[0019] 1.3 The mouse lungs were isolated, placed in a culture medium containing Dulbecco's Phosphate Buffered Saline (DPBS) for washing, the blood was washed away and the connective tissue was cut off;

[0020] 1.4 The washed lung tissue was evenly minced in a petri dish, and the fragments should be less than 1 mm 3 ;

[0021] 1.5 The tissue fragments were transferred to a 15 ml centrifuge tube containing 5 ml of dissociation solution, incubated in a water bath at 37 °C for 30 min, taken out and mixed at regular intervals until digestion was complete;

[0022] 1.6 Filter the suspension in the centrifuge tube through a 70-μm filter into a new 15-ml centrifuge tube, and add 5 ml of high-glucose culture medium (Dulbecco’s modified Eagle’s medium, DMEM) containing 10% FBS to terminate digestion;

[0023] 1.7 Under room temperature conditions, centrifuge at a relative centrifugal force of 300 g for 5 minutes;

[0024] 1.8 After discarding the supernatant, add 3 ml of 40% Percoll cell separation solution to the cell pellet and mix well. Transfer it to a centrifuge tube containing 3 ml of 70% percoll at the bottom, and centrifuge at a relative centrifugal force of 800 g for 20 min at room temperature;

[0025] 1.9 After centrifugation, the liquid is layered. Take the middle cloudy liquid, add DMEM for washing, and centrifuge at 300 g for 5 min at room temperature; repeat 2 - 3 times, discard the supernatant, and obtain pure mouse lung immune cells.

[0026] As an improvement of the above technical solution, in step 1.8, after discarding the supernatant containing lung cell debris, first add 3 ml of red blood cell lysate to the pellet and lyse for 10 min to completely remove red blood cells; then add 5 ml of PBS to terminate the red blood cell lysis procedure, and centrifuge at a relative centrifugal force of 400 g under 4℃ conditions.

[0027] As an improvement of the above technical solution, the preparation method of the dissociation solution is: 0.1% collagenase IV and 0.02% DNase I, and the volume of the dissociation solution is 6 times or more of the tissue volume.

[0028] As an improvement of the above technical solution, the metal isotope uses a Cd metal isotope, the antibody conjugation kit uses the Maxpar MCP9 kit, and S2 includes the following steps:

[0029] 2.1 Take a tube, resuspend the polymer with 87 μl of L-Buffer, and add 13 μl of 50 mM Cd element solution. Incubate in a water bath at 37℃ for 60 min, transfer it to a 3-kD column containing 100 μl of L-buffer, then add 100 μl of L-Buffer to the polymer tube to wash the tube wall, and then transfer it to a 3-kDa filter column, mix well, centrifuge at 12000 g for 25 min at room temperature, discard the supernatant, add 300 μl of L-Buffer, centrifuge at 12000 g for 30 min at room temperature, add 400 μl of C-Buffer, and centrifuge at 12000 g for 45 min at room temperature;

[0030] 2.2 Add 100 μg of antibody to a 50 kDa column, make up the volume to 400 μl with R buffer, centrifuge at 12,000 g for 10 min at room temperature, discard the waste liquid, add 400 μl of R-Buffer, centrifuge at 12,000 g for 10 min at room temperature, and repeat once;

[0031] 2.3 Dilute TCEP with R-buffer to a final concentration of 4 mM, take 100 μl and add it to the 50 kDa column containing the antibody, and incubate in a water bath at 37 °C for 30 min;

[0032] 2.4 Add 400 μl of C-buffer to a 3 kD column, centrifuge at 12,000 g for 30 min at room temperature;

[0033] 2.5 After the water bath, take the 50 kDa column, add 300 μl of C-buffer to the filter column, centrifuge at 12,000 g for 10 min at room temperature, discard the waste liquid, and repeat the operation once;

[0034] 2.6 Resuspend the metal and polymer conjugate in the 3 kDa filter with 60 μl of C-buffer, transfer the volume to the 50 kDa filter containing the reduced antibody, with a total volume of about 100 μl, gently pipette to mix evenly, and incubate in a water bath at 37 °C for 90 min;

[0035] 2.7 After the water bath, take out the 50 kDa filter column, add 200 μl of W-Buffer to the filter element, mix well, transfer it to a newly labeled 100 kDa filter element, and centrifuge at 5000 g for 10 min at room temperature;

[0036] 2.8 Add 400 μl of W-Buffer, centrifuge at 5000 g for 10 min at room temperature, discard the waste liquid, and repeat three times;

[0037] 2.9 Add 75 μl of W-Buffer to the filter element, take 2 μl of the liquid, measure its optical density value at 280 nm, and calculate the antibody concentration;

[0038] 2.10 Centrifuge at 12,000 g for 5 min at room temperature, add the volume of the antibody stabilizing solution, and obtain the metal-labeled antibody.

[0039] As an improvement of the above technical solution, the cd metal isotope includes 111Cd, 112Cd, 113Cd, 114Cd, 116Cd.

[0040] As an improvement of the above technical solution, the metal isotope uses lanthanide metal, the antibody conjugation kit uses the Maxpar X8 kit, and the S2 includes the following steps:

[0041] 2.1 Take the tube, add 95 ul of L-Buffer to resuspend the polymer, and add 5 ul of 50 mM lanthanide metal solution, mix well, incubate in a water bath at 37 °C for 30 min, transfer it to a 3 kD column containing 200 ul of L-buffer, mix well, centrifuge at 12000 g at room temperature for 25 min, discard the supernatant, and repeat once;

[0042] 2.2 Take a 50 kDa column, add 100 ug of antibody, make up the volume to 400 ul with Rbuffer, centrifuge at 12000 g at room temperature for 10 Min;

[0043] 2.3 Dilute TCEP with R-buffer to a final concentration of 4 mM, take 100 ul and add it to the 50 kDa column containing the antibody, incubate in a water bath at 37 °C for 30 min;

[0044] 2.4 Add 400 ul of C-buffer to the 3 kD column, centrifuge at 12000 g at room temperature for 30 min;

[0045] 2.5 After the water bath, take the 50 kDa column, add 300 ul of C-buffer to the filter column, centrifuge at 12000 g at room temperature for 10 min, discard the waste liquid, and repeat once;

[0046] 2.6 Resuspend the metal-polymer conjugate in the 3 kDa filter with 60 ul of C-buffer, transfer it to the 50 kDa filter containing the reduced antibody, with a total volume of about 100 ul, gently pipette to mix well, incubate in a water bath at 37 °C for 90 min;

[0047] 2.7 After the water bath, take out the 50 kDa filter column, add 200 ul of W-Buffer to the filter, centrifuge at 12000 g at room temperature for 10 min;

[0048] 2.8 Add 400 ul of W-Buffer, centrifuge at 12000 g at room temperature for 10 min, discard the waste liquid, and repeat three times;

[0049] 2.9 Add 80 ul of W-Buffer to the filter, take 2 ul of the liquid, measure its absorbance at 280 nm, and calculate the antibody concentration;

[0050] 2.10 Centrifuge at 12000 g at room temperature for 10 min, add the volume of the antibody stabilizing solution, and obtain the metal-labeled antibody.

[0051] As an improvement to the above technical solution, the lanthanide metal isotopes include: 89Y, 166Er, 152Sm, 168Er, 160Gd, 161Dy, 167Er, 159Tb, 163Dy, 154Sm, 175Lu, 165Ho, 143Nd, 153Eu, 172Yb, 150Nd, 151Eu, 170Er, 142Nd, 144Nd, 141Pr, 164Dy, 146Nd, 171Yb, 176Yb, 174Yb, 145Nd, 156Gd, 149Sm, 158Gd, 148Nd, 147Sm, 162Dy, 173Yb, 155Gd, 169Tm.

[0052] As an improvement to the above technical solution, S3 includes the following steps:

[0053] 3.1 Take 3×106 lung immune cells extracted in S1 and resuspend them with DPBS;

[0054] 3.2 Transfer them into a flow tube, add cisplatin with a final concentration of 0.5 μM, mix well and let stand at room temperature for 2 min;

[0055] 3.3 Add 2 ml of Cell Staining Buffer to terminate the reaction, and centrifuge at 300 g for 5 min at room temperature.

[0056] 3.4 Add 100 μl of surface protein antibody cocktail to each tube of the sample, gently pipette to mix the cells, let stand at room temperature for 15 min, gently mix the cells again, and continue to let stand at room temperature for 15 min;

[0057] 3.5 Add 2 ml of Cell Staining Buffer to each tube of the sample, centrifuge at 300 g for 5 min at room temperature, discard the supernatant, and repeat once. Gently mix well and disperse the cells in the remaining supernatant;

[0058] 3.6 Add 1 ml of 1X Fix I solution to each tube, mix well. Let stand at room temperature for 15 min;

[0059] 3.7 Add 2 ml of Perm-S buffer to each tube, centrifuge at 800 g for 5 min, discard the supernatant, and repeat once;

[0060] 3.8 Add 100 μl of intracellular protein and nuclear protein antibody cocktail to each tube of the sample, gently pipette to mix the cells, let stand at room temperature for 15 min, gently mix the cells again, and continue to let stand at room temperature for 15 min, and repeat step 3.5;

[0061] 3.9 Add 1 ml of 1.6% formaldehyde solution to each sample, mix well, let stand at room temperature for 10 min, centrifuge at 800 g for 5 min at room temperature, and discard the supernatant;

[0062] 3.10 Add 1 ml of cell intercalation solution to the sample, incubate at room temperature for 1 hour or overnight at 4 °C;

[0063] 3.11 Centrifuge at 800 g for 5 min at room temperature, discard the supernatant, resuspend the cells with 2 ml of Cell Staining Buffer, centrifuge at 800 g for 5 min at room temperature, and discard the supernatant.

[0064] 3.12 Resuspend the cells with 1 ml of CellAcquisition Solution, take 10 μl for cell counting. Centrifuge the remaining suspension at 800 g for 5 min at room temperature, and discard the supernatant. Place the cell pellet on ice until resuspended before loading onto the machine.

[0065] 3.13 According to the cell counting results, before loading onto the machine, resuspend the cells with CellAcquisition Solution to a suspension of 1×10 6 / ml.

[0066] 3.14 Add 10% EQ Beads to the cell suspension, filter the cell suspension through a flow tube with a 35-μm filter, and collect data on the machine.

[0067] Advantages of the present invention:

[0068] 1. In the present invention, the lung tissue is evenly minced and dissociated, centrifuged according to the cell density, and a large number of immune cells are separated. Washing is carried out using a culture medium, which maximally ensures the yield and activity of lung immune cells. The viability of the obtained cells can be known through trypan blue staining, and it is basically greater than 90%.

[0069] 2. The MaxPAR X8 and MaxparMCP9 antibody conjugation kits are commercial products, which only contain metal isotopes and conjugation reagents. The present invention uses this kit to connect stable metal isotopes with commercial purified antibody products, and at the same time designs a mass spectrometry flow cytometry channel based on the principle of minimizing channel interference, so as to comprehensively describe the classification and functions of all immune cells in the mouse lung.

[0070] 3. If traditional flow cytometry detection technology is used for detection, it has high requirements for technicians, requires a relatively large sample volume, and there is fluorescence laser spectral overlap, resulting in a relatively high background. The present invention can simultaneously detect 42 antibodies for mouse lung immune cells, detect cell-specific markers and functional proteins, and observe the dynamic changes of lung cell subsets. Description of the drawings

[0071] Figure 1 TSN map of pulmonary immune cells in mice with acute lung injury;

[0072] Figure 2 Marker expression of each population of immune cells in the lungs of mice with acute lung injury. Detailed implementation mode

[0073] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0074] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there may also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time.

[0075] Embodiment

[0076] The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury in this embodiment includes the following steps:

[0077] S1 Extract immune cells

[0078] Take mice with acute lung injury caused by intraperitoneal injection of Daboia russelii venom, sacrifice them at 6 h, perform cardiac perfusion, remove the blood inside the lung tissue after perfusion, and then remove the lungs; Cut the lungs into pieces and mix them with dissociation solution, digest in a water bath for 30 min, and then perform density gradient centrifugation and red blood cell lysis to obtain pure pulmonary immune cells of mice;

[0079] 1.1 Take mice with acute lung injury caused by intraperitoneal injection of Daboia russelii venom, sacrifice them at 6 h, wipe with 75% alcohol cotton, and cut open the chest;

[0080] 1.2 Perform cardiac perfusion, continuously perfuse normal saline through the heart, and remove the internal blood to change the lung tissue from blood red to white;

[0081] 1.3 Isolate the lungs of mice, place them in a medium containing DPBS for washing, wash away the blood and cut off the connective tissue;

[0082] 1.4 Place the washed lung tissue in a culture dish and cut it into pieces evenly. The pieces should be less than 1 mm 3 ;

[0083] 1.5 Transfer the tissue fragments into a 15 ml centrifuge tube containing 5 ml of dissociation solution, place in a 37°C water bath for 30 min, take out and mix at regular intervals until digestion is complete;

[0084] 1.6 Filter the suspension in the centrifuge tube through a 70um filter into a new 15ml centrifuge tube, and add 5ml of DMEM containing 10% FBS to terminate digestion;

[0085] 1.7 Centrifuge at 300 g for 5 minutes at room temperature;

[0086] 1.8 After discarding the supernatant, add 3 ml of 40% Percoll cell separation solution to the cell pellet and mix well, transfer to a centrifuge tube containing 3 ml of 70% Percoll at the bottom, and centrifuge at room temperature at a relative centrifugal force of 800g for 20 minutes; if there are many red blood cells in the extracted cells, discard the supernatant containing lung cell fragments, first add 3 ml of red blood cell lysis solution to the pellet for 10 minutes to completely remove the red blood cells; then add 5 ml of PBS to terminate the red blood cell lysis procedure, and centrifuge at 4°C at a relative centrifugal force of 400g;

[0087] 1.9 After centrifugation, the liquid was separated into layers. The cloudy liquid in the middle was taken and washed with DMEM. The mixture was centrifuged at room temperature at 300 g for 5 min. This process was repeated 2-3 times. The supernatant was discarded to obtain pure mouse lung immune cells.

[0088] S2 coupling

[0089] Using an antibody coupling kit, metal isotopes were linked to mouse lung immune cell marker antibodies to obtain metal-labeled antibodies;

[0090] Labeling of mass spectrometry flow antibodies (Maxpar MCP9 kit for CD metals and MaxparX8 kit for other lanthanide metals) The following is the operation process using the MaxparX8 kit

[0091] 2.1 Take the tube and add 95ul L-Buffer to resuspend the polymer, and add 5ul 50mM lanthanide metal solution to mix well, incubate at 37℃ for 30min, transfer it to a 3kD column filled with 200ul L-buffer, mix well, centrifuge at room temperature at 12000g for 25min, discard the supernatant, and repeat once;

[0092] 2.2 Take a 50kDa column and add 100ug of antibody, add Rbuffer to make up the volume to 400ul, and centrifuge at room temperature at 12000g x 10

[0093] Min;

[0094] 2.3 Dilute TCEP to a final concentration of 4 mM with R-buffer. Take 100 ul and add it to the 50 kDa column containing the antibody. Incubate in a water bath at 37 °C for 30 min;

[0095] 2.4 Add 400 ul of C-buffer to the 3 kD column and centrifuge at 12,000 g at room temperature for 30 min;

[0096] 2.5 After the water bath, take the 50 kDa column. Add 300 ul of C-buffer to the filter column and centrifuge at 12,000 g at room temperature for 10 min. Discard the waste liquid and repeat once;

[0097] 2.6 Resuspend the metal and polymer conjugate in the 3 kDa filter with 60 ul of C-buffer, transfer it to the 50 kDa filter containing the reduced antibody. The total volume is about 100 ul. Gently pipette to mix well and incubate in a water bath at 37 °C for 90 min;

[0098] 2.7 After the water bath, take out the 50 kDa filter column. Add 200 ul of W-Buffer to the filter and centrifuge at 12,000 g at room temperature for 10 min;

[0099] 2.8 Add 400 ul of W-Buffer, centrifuge at 12,000 g at room temperature for 10 min, discard the waste liquid and repeat three times;

[0100] 2.9 Add 80 ul of W-Buffer to the filter. Take 2 ul of the liquid and measure its absorbance at 280 nm to calculate the antibody concentration;

[0101] 2.10 Centrifuge at 12,000 g at room temperature for 10 min and add the volume of the antibody stabilizing solution to obtain the metal-labeled antibody.

[0102] The following is the operation procedure for using the Maxpar MCP9 kit:

[0103] 2.1 Take a tube and resuspend the polymer with 87 ul of L-Buffer and 13 ul of 50 mM Cd element solution. Incubate in a water bath at 37 °C for 60 min. Transfer it to the 3 kD column containing 100 ul of L-buffer. Then add 100 ul of L-Buffer to wash the tube wall and transfer it to the 3 kDa filter column. Mix well and centrifuge at 12,000 g at room temperature for 25 min. Discard the supernatant. Add 300 ul of L-Buffer and centrifuge at 12,000 g at room temperature for 30 min. Add 400 ul of C-Buffer and centrifuge at 12,000 g at room temperature for 45 min;

[0104] 2.2 Add 100 μg of antibody to a 50 kDa column, and make up the volume to 400 μl with R buffer. Centrifuge at 12,000 g at room temperature for 10 min, discard the waste liquid, add 400 μl of R-Buffer, centrifuge at 12,000 g at room temperature for 10 min, and repeat once.

[0105] 2.3 Dilute TCEP with R-buffer to a final concentration of 4 mM. Take 100 μl and add it to the 50 kDa column containing the antibody, and incubate in a water bath at 37 °C for 30 min.

[0106] 2.4 Add 400 μl of C-buffer to a 3 kDa column and centrifuge at 12,000 g at room temperature for 30 min.

[0107] 2.5 After the water bath, take the 50 kDa column, add 300 μl of C-buffer to the filter column, centrifuge at 12,000 g at room temperature for 10 min, discard the waste liquid, and repeat the operation once.

[0108] 2.6 Resuspend the metal and polymer conjugate in the 3 kDa filter with 60 μl of C-buffer, transfer it to the 50 kDa filter containing the reduced antibody, with a total volume of about 100 μl. Gently pipette to mix well and incubate in a water bath at 37 °C for 90 min.

[0109] 2.7 After the water bath, take out the 50 kDa filter column, add 200 μl of W-Buffer to the filter, mix well, transfer it to a newly labeled 100 kDa filter, and centrifuge at 5,000 g at room temperature for 10 min.

[0110] 2.8 Add 400 μl of W-Buffer, centrifuge at 5,000 g at room temperature for 10 min, discard the waste liquid, and repeat three times.

[0111] 2.9 Add 75 μl of W-Buffer to the filter. Take 2 μl of the liquid and measure its optical density at 280 nm to calculate the antibody concentration.

[0112] 2.10 Centrifuge at 12,000 g at room temperature for 5 min, and add the volume of the antibody stabilizing solution to obtain the metal-labeled antibody.

[0113] 2.10 Centrifuge at room temperature, 12,000 g X 5 min, add the volume of the antibody stabilizing solution, and obtain the metal-labeled antibody.

[0114] The metal isotopes, R buffer, L-Buffer, C-Buffer, and W-Buffer reagents involved in the above steps in the present invention

[0115] are all from the MaxPAR X8 and MaxPAR MCP9 antibody conjugation kits (Fluidigm, America).

[0116] The metal isotopes involved in the present invention include 89Y, 166Er, 152Sm, 168Er, 160Gd, 161Dy, 167Er, 159Tb, 163Dy, 154Sm, 175Lu, 165Ho, 143Nd, 153Eu, 172Yb, 150Nd, 151Eu, 170Er, 209Bi, 142Nd, 144Nd, 141Pr, 164Dy, 146Nd, 171Yb, 176Yb, 116Cd, 174Yb, 113Cd, 111Cd, 114Cd, 145Nd, 156Gd, 149Sm, 158Gd, 148Nd, 147Sm, 162Dy, 173Yb, 112Cd, 155Gd, 169Tm.

[0117] The antibodies involved in the present invention include anti-CD45, anti-CD19, anti-CD3e, anti-CD8a, anti-CD62L, anti-T-bet, anti-GATA-3, anti-ROR gamma / t, anti-Bcl6, anti-CTLA-4, anti-CD127, anti-Foxp3, anti-CD69, anti-CD335 / NKp46, anti-CD11b, anti-CD27, anti-CD64, anti-CD169 / Siglec-1, anti-I-A / I-E / MHC class II, anti-CD11c, anti-CD115, anti-Ly6G, anti-CX3CR1, anti-CD43, anti-CD38, anti-FceR1a, anti-B220, anti-CD5, anti-CD138, anti-CD4, anti-CD44, anti-TCRgd, anti-KLRG1, anti-CD25, anti-CD49b, anti-MerTK, anti-F4 / 80, anti-CD103, anti-CD24, anti-Ly6C, anti-CCR2, anti-Ki67.

[0118] S3 staining

[0119] Incubate the isolated mouse lung immune cells with the metal-labeled antibody to label the immune cells;

[0120] 3.1 Take 3X10 6 lung immune cells extracted in step (1) and resuspend them with DPBS;

[0121] 3.2 Transfer it into a flow tube, add cisplatin with a final concentration of 0.5 μM, mix well and leave it at room temperature for 2 min;

[0122] 3.3 Add 2 ml of Cell Staining Buffer (CSB) to terminate the reaction, and centrifuge at 300 g for 5 min at room temperature.

[0123] 3.4 Add 100 μl of surface protein antibody cocktail to each tube of the sample, gently pipette to mix the cells, leave it at room temperature for 15 min, gently mix the cells again, and continue to leave it at room temperature for 15 min;

[0124] 3.5 Add 2 ml of Cell Staining Buffer to each tube of the sample, centrifuge at 300 g for 5 min at room temperature, discard the supernatant, and repeat once. Gently mix well and disperse the cells in the remaining supernatant;

[0125] 3.6 Add 1 ml of 1X Fix I solution to each tube, mix well. Leave it at room temperature for 15 min;

[0126] 3.7 Add 2 ml of Perm-S buffer to each tube, centrifuge at 800 g for 5 min, discard the supernatant, and repeat once;

[0127] 3.8 Add 100 μl of intracellular protein and nuclear protein antibody cocktail to each tube of the sample, gently pipette to mix the cells, leave it at room temperature for 15 min, gently mix the cells again, and continue to leave it at room temperature for 15 min, and repeat step 3.5;

[0128] 3.9 Add 1 ml of 1.6% formaldehyde solution to each sample, mix well, leave it at room temperature for 10 min, centrifuge at 800 g for 5 min at room temperature, and discard the supernatant;

[0129] 3.10 Add 1 ml of cell intercalation solution to the sample, leave it at room temperature for 1 hour or overnight at 4 °C;

[0130] 3.11 Centrifuge at 800 g for 5 min at room temperature, discard the supernatant, add 2 ml of Cell Staining Buffer to resuspend the cells, centrifuge at 800 g for 5 min at room temperature, and discard the supernatant.

[0131] 3.12 Add 1 ml of Cell Acquisition Solution to resuspend the cells, take 10 μl for cell counting. Centrifuge the remaining suspension at 800 g for 5 min at room temperature, and discard the supernatant. Place the cell pellet on ice until resuspended before loading onto the machine.

[0132] 3.13 According to the cell counting results, before loading onto the machine, resuspend the cells with CellAcquisition Solution to a suspension of 1X106 / ml.

[0133] 3.14 Add 10% EQ Beads to the cell suspension, filter the cell suspension with a flow tube with a 35um filter mesh, and collect data on the machine.

[0134] The preparation method of the Antibody Cocktail described in the present invention is as follows: Take the antibody and add and mix it according to the dilution multiple, quantitatively add Cell Staining Buffer to 100ul, and mix well to obtain the Antibody Cocktail.

[0135] The preparation method of the cell intercalatio described in the present invention is as follows: Add Ir with a final concentration of 125nM or Rh with a final concentration of 500nM to the fix and perm buffer, and the dosage for each sample is 1ml.

[0136] S4 Bioinformatics analysis

[0137] The labeled mouse lung immune cells are analyzed on a mass cytometry machine. The t-SNE and phenograph algorithms are used to perform dimensionality reduction and clustering on the obtained data; then, the expression distributions of multiple detection antibodies of different cell subsets are displayed on the same heat map, and the expression levels of different markers in different cell subsets and the proportion of cell subsets in the total number of cells are shown through the t-SNE map, so as to show the classification and functional observation of mouse lung immune cells.

[0138] Since the antibody labeled with a metal element recognizes and binds to the antigen on the cell surface or inside, the cells with the antibody labeled with a metal element are sent into the plasma torch one by one for ionization, so that metal ions are released. The released metal ions are sent into the time-of-flight detection chamber for separation and detection. The detector will accurately record the time when various ions arrive, and then calculate the accurate content of various metal tags in each cell, so as to obtain the antigen expression level on the cell surface or inside. Then, dimensionality reduction processing is performed, and the t-SNE and phenograph are used to analyze the data of mass cytometry. The expression distributions of 42 detection antibodies of different cell subsets are on one heat map, and the expression of different markers and the distribution of different cell subsets are shown through the viSNE map.

[0139] Figure 1 Among them, the distribution of different shades of colors represents the distribution of different cell subsets, and the size represents the abundance of cell subsets (the numbers in the figure refer to the cell subsets obtained according to dimensionality reduction analysis, corresponding to the heat map, and are not used as reference numerals in the present invention, so they will not be described one by one). Figure 2Among them, the color blocks of different colors represent the expression distribution of 42 antibodies in different cell subsets.

[0140] Based on the expression of cell surface markers, we obtained 32 cell subsets. CD45+ cells are the total immune cells, which can be divided into the following subsets:

[0141] (1) B cells: CD19+, B220+, CD5+;

[0142] (2) Plasma cells: CD19+, B220+, CD138+;

[0143] (3) CD4+ T cells: CD3+, CD4+, CD8-, which can be further divided into central memory T (Tcm) cells (CD44hi, CD62Lhi) and effector memory T (Tem) cells (CD44hi, CD62Llo). Tem can be further divided into T-bet+ Th1, GATA3+ Th2, RORyt+ Th17, Bcl6+ Tfh;

[0144] (4) CD8+ T cells: CD3+, CD4-, CD8+. Naive CD8 T cells (Tn) are CD44lo, CD62Lhi, and Teff cells are CD44hi, CD62Llo. Teff cells can be further divided into short-lived effector cells (SLEC: CD127-, KLRG1+) and memory precursor effector cells (MPEC: CD127+, KLRG1-);

[0145] (5) Regulatory T cells (Treg): CD8-, CD4+, CD25+, Foxp3+;

[0146] (6) γδ T cells: CD3+, TCRβ+;

[0147] (7) Tissue-resident T cells (Trm): CD8+, CD44hi, CD62Llo, CD69hi;

[0148] (8) NK cells: CD49b+, NKp46+. Immature NK cells are expressed as CD11blow, CD27high, then mature to express double-positive CD27+ CD11b+, and fully mature to express CD27low CD11bhigh;

[0149] (9) NKT cells: CD3+, NKp46+;

[0150] (10) Macrophages can be divided into alveolar macrophages (CD64+, MerTK+, SiglecF+, CD11b-, F4 / 80+) and interstitial macrophages (CD64+, MerTK+, SiglecF-, CD11b+, F4 / 80+);

[0151] (1) Dendritic cells: CD64-, MerTK-, MHCII+, CD11chi, CD11b+, and can be further divided into cCD1 (CD103+, CD11b-) and cCD2 (CD103-, CD11b+, CD24+);

[0152] (12) Monocytes: CD115+, CD11b+, Ly6C lo / hi, CD11c-, and can be further divided into patrolling monocytes (CX3CR1int, CCR2+, CD62L+, CD43lo, Ly6Chi) and inflammatory monocytes (CX3CR1hi, CCR2-, CD62L-, CD43hi, Ly6Clo);

[0153] (13) Neutrophils: CD11b+, Ly-6C lo / neg, Ly6G+;

[0154] (14) Eosinophils: CD11b+, Siglec-F, MHCII-;

[0155] (15) Basophils: Ly6Glow, Ly-6Clow, FceRIa+;

[0156]

[0157]

[0158] It should be noted that in this article, if there are relational terms such as first and second, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the said element.

[0159] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Method for establishing the characteristics of the pulmonary immune cell atlas in mice with snake venom-induced acute lung injury, which is characterized in that: It includes the following steps: S1 Extract immune cells Take mice with acute lung injury caused by intraperitoneal injection of Daboia russelii snake venom, sacrifice them at 6 h, perform cardiac perfusion, remove the blood inside the lung tissue by perfusion, and then remove the lungs; Cut the lungs into pieces, mix them with dissociation solution, digest in a water bath for 30 min, and then perform density gradient centrifugation and red blood cell lysis to obtain pure pulmonary immune cells of mice; S2 Coupling Use an antibody coupling kit to connect a metal isotope with an antibody against a pulmonary immune cell marker of mice to obtain a metal-labeled antibody; S3 Staining Incubate the isolated pulmonary immune cells of mice with the metal-labeled antibody to label the immune cells; S4 Bioinformatics analysis Analyze the labeled pulmonary immune cells of mice by mass cytometry. Use the t-SNE and phenograph algorithms to perform dimensionality reduction and clustering on the obtained data; Then display the expression distributions of multiple detection antibodies in different cell subsets on the same heat map, and show the expression levels of different markers in different cell subsets and the proportion of cell subsets in the total number of cells through the t-SNE map, so as to show the classification and functional observation of pulmonary immune cells in mice; The metal isotope uses a cd metal isotope, the antibody coupling kit uses a Maxpar MCP9 kit, and the S2 includes the following steps: 2.1 Take a tube, add 87 ul of L-Buffer to resuspend the polymer, add 13 ul of 50 mM Cd element solution, incubate in a water bath at 37 °C for 60 min, transfer it to a 3 kDa column containing 100 ul of L-Buffer, then add 100 ul of L-Buffer to the polymer tube to wash the tube wall, and then transfer it to a 3 kDa filter column, mix well, centrifuge at a relative centrifugal force of 12000 g at room temperature for 25 minutes, discard the supernatant, add 300 ul of L-Buffer, centrifuge at a relative centrifugal force of 12000 g at room temperature for 30 minutes, add 400 ul of C-Buffer, and centrifuge at a relative centrifugal force of 12000 g at room temperature for 45 minutes; 2.2 Take a 50 kDa column, add 100 ug of antibody, make up the volume to 400 ul with R-Buffer, centrifuge at a relative centrifugal force of 12000 g at room temperature for 10 minutes, discard the waste liquid, add 400 ul of R-Buffer, centrifuge at a relative centrifugal force of 12000 g at room temperature for 10 minutes, and repeat this operation once; 2.3 Dilute TCEP with R-buffer to a final concentration of 4 mM, take 100 ul and add it to the 50 kDa column containing the antibody, and incubate in a water bath at 37 °C for 30 min; 2.4 Add 400 ul of C-Buffer to the 3 kDa column, and centrifuge at a relative centrifugal force of 12000 g at room temperature for 30 minutes; 2.5 After the water bath, take the 50 kDa column, add 300 ul of C-Buffer to the filter column, centrifuge at 12,000 g relative centrifugal force for 10 minutes at room temperature, discard the waste liquid, and repeat this operation once; 2.6 Resuspend the metal and polymer conjugate in the 3 kDa filter with 60 ul of C-Buffer, transfer it to the 50 kDa filter containing the reduced antibody, with a total volume of about 100 ul, gently pipette to mix, and incubate in a water bath at 37 °C for 90 min; 2.7 After the water bath, take out the 50 kDa filter column, add 200 ul of W-Buffer to the filter, mix well, transfer it to a newly labeled 100 kDa filter, and centrifuge at 5,000 g relative centrifugal force for 10 minutes at room temperature; 2.8 Add 400 ul of W-Buffer, centrifuge at 5,000 g relative centrifugal force for 10 minutes at room temperature, discard the waste liquid, and repeat this operation three times; 2.9 Add 75 ul of W-Buffer to the filter, take 2 ul of the liquid, measure its optical density at 280 nm, and calculate the antibody concentration; 2.10 Centrifuge at 12,000 g relative centrifugal force for 5 minutes at room temperature, add the volume of the antibody stabilizing solution to obtain the metal-labeled antibody; Or, the metal isotope is a lanthanide metal, the antibody conjugation kit is the Maxpar X8 kit, and S2 includes the following steps: 2.1 Take a tube, resuspend the polymer with 95 ul of L-Buffer, add 5 ul of 50 mM lanthanide metal solution, mix well, incubate in a water bath at 37 °C for 30 min, transfer it to a 3 kD column containing 200 ul of L-Buffer, mix well, centrifuge at 12,000 g relative centrifugal force for 25 minutes at room temperature, discard the supernatant, and repeat this operation once; 2.2 Take a 50 kDa column, add 100 ug of antibody, make up the volume to 400 ul with R-Buffer, and centrifuge at 12,000 g relative centrifugal force for 10 minutes at room temperature; 2.3 Dilute TCEP with R-Buffer to a final concentration of 4 mM, take 100 ul and add it to the 50 kDa column containing the antibody, and incubate in a water bath at 37 °C for 30 min; 2.4 Add 400 ul of C-Buffer to the 3 kD column, and centrifuge at 12,000 g relative centrifugal force for 30 minutes at room temperature; 2.5 After the water bath, take the 50 kDa column, add 300 ul of C-Buffer to the filter column, centrifuge at 12,000 g relative centrifugal force for 10 minutes at room temperature, discard the waste liquid, and repeat this operation once; 2.6 Resuspend the metal and polymer conjugate in the 3 kDa filter with 60 ul of C-Buffer, transfer it to the 50 kDa filter containing the reduced antibody, with a total volume of about 100 ul, gently pipette to mix, and incubate in a water bath at 37 °C for 90 min; 2.7 After the water bath, take out the 50 kDa filter column, add 200 ul of W-Buffer to the filter element, and centrifuge at a relative centrifugal force of 12,000 g for 10 minutes at room temperature; 2.8 Add 400 ul of W-Buffer, centrifuge at a relative centrifugal force of 12,000 g for 10 minutes at room temperature, discard the waste liquid, and repeat this operation three times; 2.9 Add 80 ul of W-Buffer to the filter element, take 2 ul of the liquid, measure its absorbance at 280 nm, and calculate the antibody concentration; 2.10 Centrifuge at a relative centrifugal force of 12,000 g for 10 minutes at room temperature, and add the volume of the antibody stabilizing solution to obtain the metal-labeled antibody.

2. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 1, characterized in that: The S1 includes the following steps: 1.1 Take mice with acute lung injury caused by intraperitoneal injection of Daboia russelii snake venom, sacrifice them at 6 h, wipe them with 75% alcohol cotton, and cut open the chest; 1.2 Perform cardiac perfusion, continuously perfuse physiological saline through the heart to remove the internal blood and turn the lung tissue from blood red to white; 1.3 Isolate the mouse lungs, place them in a medium containing DPBS for washing, wash away the blood and cut off the connective tissue; 1.4 Place the cleaned lung tissue in a petri dish and cut it into small pieces evenly. The pieces should be less than 1 mm 3 ; 1.5 Transfer the tissue fragments into a 15 ml centrifuge tube containing 5 ml of dissociation solution, incubate in a water bath at 37 °C for 30 min, take it out and mix it at regular intervals until digestion is complete; 1.6 Filter the suspension in the centrifuge tube through a 70 um filter into a new 15 ml centrifuge tube, and add 5 ml of DMEM containing 10% FBS to terminate digestion; 1.7 Centrifuge at a relative centrifugal force of 300 g for 5 minutes at room temperature; 1.8 After discarding the supernatant, add 3 ml of 40% Percoll cell separation solution to the cell pellet and mix well, transfer it to a centrifuge tube containing 3 ml of 70% percoll cell separation solution at the bottom, and centrifuge at a relative centrifugal force of 800 g for 20 min at room temperature; 1.9 After centrifugation, the liquid is layered, take the middle cloudy liquid, wash it with DMEM, and centrifuge at a relative centrifugal force of 300 g for 5 minutes at room temperature; repeat this operation 2-3 times, discard the supernatant, and obtain pure mouse pulmonary immune cells.

3. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 2, characterized in that: In step 1.8, after discarding the supernatant containing lung cell fragments, first add 3 ml of red blood cell lysate to the pellet and lyse for 10 min to completely remove red blood cells; then add 5 ml of PBS to terminate the red blood cell lysis program, and centrifuge at a relative centrifugal force of 400 g at 4 °C.

4. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 1, characterized in that: The preparation method of the dissociation solution is: 0.1% collagenase IV and 0.02% DNase I, and the volume of the dissociation solution is 6 times or more of the tissue volume.

5. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 1, It is characterized in that: The Cd metal isotope is 111Cd, 112Cd, 113Cd, 114Cd or 116Cd.

6. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 1, It is characterized in that: The lanthanide metal isotopes are: 89Y, 166Er, 152Sm, 168Er, 160Gd, 161Dy, 167Er, 159Tb, 163Dy, 154Sm, 175Lu, 165Ho, 143Nd, 153Eu, 172Yb, 150Nd, 151Eu, 170Er, 142Nd, 144Nd, 141Pr, 164Dy, 146Nd, 171Yb, 176Yb, 174Yb, 145Nd, 156Gd, 149Sm, 158Gd, 148Nd, 147Sm, 162Dy, 173Yb, 155Gd or 169Tm.

7. The method for establishing the characteristics of the pulmonary immune cell atlas of mice with snake venom-induced acute lung injury according to claim 1, It is characterized in that: The S3 includes the following steps: 3.1 Take 3×10 6 lung immune cells extracted from S1 and resuspend them with DPBS; 3.2 Transfer it into a flow tube, add cisplatin with a final concentration of 0.5 μM, mix well and place it at room temperature for 2 min; 3.3 Add 2 ml of cell staining buffer to terminate the reaction, and centrifuge at a relative centrifugal force of 300 g at room temperature for 5 minutes; 3.4 Add 100 μl of surface protein antibody mixture to each tube of sample, gently pipette to mix the cells, place at room temperature for 15 min, gently mix the cells again, and continue to place at room temperature for 15 min; 3.5 Add 2 ml of cell staining buffer to each tube of sample, centrifuge at a relative centrifugal force of 300 g at room temperature for 5 minutes, discard the supernatant, and repeat this operation once; gently mix well and disperse the cells in the remaining supernatant; 3.6 Add 1 ml of 1X Fix I solution to each tube, mix well; place at room temperature for 15 min; 3.7 Add 2 ml of Perm-S Buffer to each tube, centrifuge at a relative centrifugal force of 800 g for 5 minutes, discard the supernatant, and repeat this operation once; 3.8 Add 100 μl of intracellular protein and nuclear protein antibody mixture to each tube of sample, gently pipette to mix the cells, place at room temperature for 15 min, gently mix the cells again, and continue to place at room temperature for 15 min, and repeat step 3.5; 3.9 Add 1 ml of 1.6% formaldehyde solution to each sample, mix well, place at room temperature for 10 min, centrifuge at a relative centrifugal force of 800 g at room temperature for 5 minutes, and discard the supernatant; 3.10 Add 1 ml of cell intercalation solution to the sample, at room temperature for 1 hour or overnight at 4 °C; 3.11 Centrifuge at a relative centrifugal force of 800 g at room temperature for 5 minutes, discard the supernatant, add 2 ml of cell staining buffer to resuspend the cells, centrifuge at a relative centrifugal force of 800 g at room temperature for 5 minutes, and discard the supernatant; 3.12 Resuspend the cells by adding 1 ml of cell harvesting solution, and take 10 μl for cell counting; centrifuge the remaining suspension at 800 g relative centrifugal force for 5 minutes at room temperature, discard the supernatant; place the cell pellet on ice until resuspended immediately before loading onto the instrument. 3.13 According to the cell counting results, before loading onto the instrument, resuspend the cells with cell acquisition solution to a suspension of 1×10 6 / ml; 3.14 Add 10% calibration microspheres to the cell suspension, filter the cell suspension through a flow tube with a 35-μm filter, and collect data on the instrument.

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