CN116790500A - Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry - Google Patents
Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry Download PDFInfo
- Publication number
- CN116790500A CN116790500A CN202310734868.3A CN202310734868A CN116790500A CN 116790500 A CN116790500 A CN 116790500A CN 202310734868 A CN202310734868 A CN 202310734868A CN 116790500 A CN116790500 A CN 116790500A
- Authority
- CN
- China
- Prior art keywords
- sorting
- depleted
- tumor
- cell suspension
- lung cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 57
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 46
- 238000001764 infiltration Methods 0.000 title claims abstract description 32
- 230000008595 infiltration Effects 0.000 title claims abstract description 29
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 21
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 21
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 41
- 239000006285 cell suspension Substances 0.000 claims abstract description 38
- 101001046687 Homo sapiens Integrin alpha-E Proteins 0.000 claims abstract description 30
- 102100022341 Integrin alpha-E Human genes 0.000 claims abstract description 30
- 102100029722 Ectonucleoside triphosphate diphosphohydrolase 1 Human genes 0.000 claims abstract description 27
- 101001012447 Homo sapiens Ectonucleoside triphosphate diphosphohydrolase 1 Proteins 0.000 claims abstract description 27
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims abstract description 19
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims abstract description 19
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims abstract description 18
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims abstract description 18
- 238000002347 injection Methods 0.000 claims abstract description 15
- 239000007924 injection Substances 0.000 claims abstract description 15
- 102000029816 Collagenase Human genes 0.000 claims abstract description 10
- 108060005980 Collagenase Proteins 0.000 claims abstract description 10
- 229960002424 collagenase Drugs 0.000 claims abstract description 10
- 238000011534 incubation Methods 0.000 claims abstract description 10
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims abstract description 7
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims abstract description 7
- 230000000694 effects Effects 0.000 claims abstract description 7
- 230000009274 differential gene expression Effects 0.000 claims abstract description 6
- 238000010195 expression analysis Methods 0.000 claims abstract description 6
- 238000004043 dyeing Methods 0.000 claims abstract description 5
- 238000011068 loading method Methods 0.000 claims abstract description 4
- 238000012163 sequencing technique Methods 0.000 claims abstract description 4
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 claims abstract 3
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims description 30
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 27
- 230000029087 digestion Effects 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 5
- 210000004698 lymphocyte Anatomy 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000012894 fetal calf serum Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 210000003289 regulatory T cell Anatomy 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 claims 2
- 206010064390 Tumour invasion Diseases 0.000 claims 2
- 230000009400 cancer invasion Effects 0.000 claims 2
- 229940088598 enzyme Drugs 0.000 claims 2
- 230000000779 depleting effect Effects 0.000 abstract 2
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000000926 separation method Methods 0.000 description 5
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for separating lung cancer tissue tumor infiltration depleted T lymphocytes by flow cytometry, which comprises the following steps: s1, digesting lung cancer tumor tissues by collagenase to obtain single cell suspension; s2, taking two parts of single cell suspension, wherein one part of single cell suspension is added with antibodies CD45/CD3/CD19/CD4/CD8/CD39/CD103/L-D for dyeing and then used as a sample injection component for selecting and depleting T cells, and the other part of single cell suspension is added with antibodies CD45/CD3/CD19/CD8/CD4/CD127/CD25/L-D for dyeing and then used as a control group for selecting and depleting cell population Treg; s3, loading the cell single-cell suspension after incubation of the antibody to perform cell sorting; s4, performing differential gene expression analysis on the sample injection group and the control group of the sorted tumor infiltration depleted T lymphocyte group by bulk transcriptome sequencing; the sorting method provided by the invention adopts a flow sorting technology, has lower use cost and higher clinical accessibility, sorts the rare cells with high purity and high activity by using the sorting function of the instrument, and has important scientific significance and application value.
Description
Technical Field
The invention relates to the technical field of cell detection. In particular to a method for separating lung cancer tissue tumor infiltration depleted T lymphocytes by flow cytometry.
Background
Chinese patent 201880038128.3 discloses the use of CD39 and CD103 in the identification of human tumor-reactive T cells for cancer treatment, evaluates the phenotype and function of tumor-reactive CD8T cells, and determines that co-expression of CD103 and CD39 identifies a tumor-infiltrating CD8T cell population specifically induced in the tumor microenvironment. These cells, which are stimulated chronically within the tumor, are highly enriched for tumor reactivity and have the ability to kill autologous tumor cells. Higher frequency CD8 in human tumors + CD39 + CD103 + T cells are associated with higher overall survival rates, and it has been determined that the presence of cd8+cd39+cd103+ T cells may indicate that the therapeutic approach is effective for treating tumors.
The cell sorting technology is widely applied in scientific research, can perform multiparameter comprehensive sorting according to the analysis result of the concentrated marker, and can realize sorting of different fluorescence intensities. The existing sorting products are mostly dependent on antibody magnetic beads to sort out cells carrying specific antigens, such as CD45, CD14 and the like, and are based on the fact that cell surface antigens can be combined with specific monoclonal antibodies connected with the magnetic beads, in an external magnetic field, cells connected with the magnetic beads through antibodies are adsorbed and retained in the magnetic field, and cells without the surface antigens cannot be combined with the specific monoclonal antibodies connected with the magnetic beads, but are not magnetic and retained in the magnetic field, so that the cells can be separated, but can only be sorted according to one parameter at a time, and the cells marked by the specific antigen combination cannot be sorted out at one time.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for separating lung cancer tissue tumor infiltration depleted T lymphocytes by flow cytometry, the use cost is low by adopting a flow separation technology, the clinical accessibility is high, and the rare cells with high purity and high activity are obtained by separation by utilizing the separation function of an instrument, so that the method has important scientific significance and application value.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for sorting lung cancer tissue tumor infiltration depleted T lymphocytes by flow cytometry, comprising the steps of:
s1, preparing single-cell suspension, and digesting the treated lung cancer tumor tissue by collagenase to obtain single-cell suspension;
s2, adding an antibody into a single cell suspension for incubation, taking two equal single cell suspensions according to a TILs identification scheme, wherein the antibody CD45/CD3/CD19/CD4/CD8/CD39/CD103/L-D is added into one single cell suspension, and dyeing and incubating for 15min at room temperature, and taking the single cell suspension as a sample injection component of a follow-up flow cytometer to sort and exhaust T cells (LD - CD45 + CD3 + CD19 - CD8 + CD4 - CD103 + CD39 + ) The other single cell suspension was then stained with antibody CD45/CD3/CD19/CD8/CD4/CD127/CD25/L-D at room temperature for 15min, and used as a control group for a subsequent flow cytometer to sort non-depleted cell population tregs (LD - CD45 + CD3 + CD19 - CD8 - CD4 + CD127 - CD25 + ) Waiting for on-machine detection;
s3, flow-sorting target cell groups, respectively loading sample injection group single-cell suspension after incubation of antibodies and control group single-cell suspension, analyzing by a flow cytometer, and carrying out tumor infiltration depletion on T lymphocytes (CD 8) + CD103 + CD39 + ) Setting a ring gate, adjusting the sorting angle of the flow cytometer, and sorting cells;
s4, verifying the sorting efficiency, and carrying out differential gene expression analysis on the cell population of the sample injection group and the control group of the sorted tumor infiltration depleted T lymphocyte population by bulk transcriptome sequencing.
Further, in step S1: the treatment of lung cancer tumor tissue includes: after fresh tumor tissue is collected, the fresh tumor tissue is completely soaked in RPMI+10% FBS solution at the temperature of 4 ℃ and is placed on ice and stored in an ice box;
the collagenase digestion treatment includes: rinsing fresh tissue samples stored for no more than 2 hours with 1 XPBS at 4 ℃, cutting up the tissue samples with a sterilizing scalpel blade, transferring the tissue samples to a 2mL centrifuge tube filled with a tissue digestion solution, fully and uniformly mixing the tissue samples, carrying out water bath at 37 ℃ for 1 hour, filtering the tissue samples with a 16-gauge needle and a 40um cell filter screen after complete digestion, and carrying out resuspension after centrifugation to calculate the yield and the activity.
Still further, the tissue digestion solution employs complete media+0.225mg/mL collagenase type IV.
Further, in step S2, before the flow cytometer is started, the suspension after incubation of the antibody is washed by PBS, and then put into a centrifuge, centrifuged at 500g for 5min, resuspended by PBS, filtered by a 40 μm cell sieve, and sterilized, and then used as a sample for subsequent flow cytometry, and the sample is waited for the start-up.
Further, the flow cytometer uses phosphate buffer as a sheath fluid, and washes a 1.5mL enzyme-free centrifuge tube with fetal calf serum, and places the enzyme-free centrifuge tube on a flow collection tube in a sorting bin of the flow cytometer.
Furthermore, in step S3, L-D/CD45 is used to gate out lymphocytes, and then the tumor infiltration depleted T lymphocyte population (CD 3) is obtained by sequentially gating CD19/CD3, CD8/CD4 and CD103/CD39 + CD8 + CD103 + CD39 + ) And sorted into a flow collection tube.
Further, in step S4, tumor infiltration-depleted T lymphocyte population (CD 3 + CD8 + CD103 + CD39 + ) The gene CD8A and ITGAE (encoding CD 103) are obviously expressed, and the cell population in single cell suspension of the sample injection group is verified to be tumor infiltration depletion T lymphocyte population (CD 3) + CD8 + CD103 + CD39 + )。
The technical scheme of the invention has the following beneficial technical effects:
tumor infiltration depletion T lymphocyte populations (CD 3) by flow cytometry + CD8 + CD103 + CD39 + ) The method of the (1) analyzes the gene expression characteristics of the cell, determines the tumor infiltration and exhaustion T lymphocyte group, adopts the flow separation technology, has lower use cost and higher clinical accessibility, and utilizes the separation function of the instrument to separate and obtain the rare cells with high purity and high activity, thereby having important scientific significance and application value.
Drawings
FIG. 1 is a schematic diagram showing the state of the single cell suspension preparation process of the present invention;
FIG. 2 is a flow chart showing the flow of the streaming gate strategy of the present invention;
FIG. 3 is a schematic diagram showing the results of differential gene expression analysis of tumor-infiltrating depleted T lymphocyte populations and control cell populations according to the present invention.
Detailed Description
The method for sorting lung cancer tissue tumor infiltration and depletion T lymphocyte by flow cytometry comprises the following steps:
s1, preparing single-cell suspension, and digesting the treated lung cancer tumor tissue by collagenase to obtain single-cell suspension;
the treatment of lung cancer tumor tissue includes: after fresh tumor tissue is collected, the fresh tumor tissue is completely soaked in RPMI+10% FBS solution at the temperature of 4 ℃ and is placed on ice and stored in an ice box;
the collagenase digestion treatment includes: fresh tissue samples stored for no more than 2 hours were rinsed with 1 XPBS at 4℃, minced with a sterile scalpel blade, transferred to a 2mL centrifuge tube containing a tissue digestion solution (complete media+0.225mg/mL collagenase type IV), thoroughly mixed, water-bath at 37℃ for 1 hour, filtered with a 16-gauge needle and a 40um cell screen after complete digestion, and resuspended to calculate the yield and activity after centrifugation, as shown in FIG. 1, the digested state is the state before sheared tissue was added to digestive enzymes, at this point not being a single cell suspension, except the tissue suspension, the middle sieving was removing the tissues such as fiber, collagen, etc. after digestion, obtaining a single cell suspension, and the trypan blue view was a single cell suspension cell state.
S2, adding an antibody into a single cell suspension for incubation, taking two equal single cell suspensions according to a TILs identification scheme, wherein the antibody CD45/CD3/CD19/CD4/CD8/CD39/CD103/L-D is added into one single cell suspension, and dyeing and incubating for 15min at room temperature, and taking the single cell suspension as a sample injection component of a follow-up flow cytometer to sort and exhaust T cells (LD - CD45 + CD3 + CD19 - CD8 + CD4 - CD103 + CD39 + ) The other single cell suspension was added with the antibodies CD45/CD3/CD19/CD8/CD4 +.CD127/CD25/L-D was stained and incubated at room temperature for 15min as a control group for subsequent flow cytometry to sort non-depleted cell population Treg (LD - CD45 + CD3 + CD19 - CD8 - CD4 + CD127 - CD25 + ) Waiting for on-machine detection;
before the flow cytometer is started, washing the suspension after incubation of the antibody by PBS, putting the suspension into a centrifuge, centrifuging for 5min at a centrifugal speed of 500g, re-suspending the suspension by PBS, filtering by a 40 mu m cell sieve, performing aseptic treatment, and taking the suspension as sample injection of the subsequent flow cytometer, and waiting for detection by the machine.
The flow cytometer uses phosphate buffer as sheath fluid, and washes a 1.5mL enzyme-free centrifuge tube with fetal calf serum, and places the enzyme-free centrifuge tube on a flow collection tube in a sorting bin of the flow cytometer.
S3, flow-sorting target cell groups, respectively loading sample injection group single-cell suspension after incubation of antibodies and control group single-cell suspension, analyzing by a flow cytometer, and carrying out tumor infiltration depletion on T lymphocytes (CD 8) + CD103 + CD39 + ) Setting a ring gate, adjusting the sorting angle of the flow cytometer, and sorting cells;
the L-D/CD45 is used for setting the portal loop out lymphocyte, and then the tumor infiltration depleted T lymphocyte group (CD 3) is obtained by sequentially setting the portal policy of CD19/CD3, CD8/CD4 and CD103/CD39 + CD8 + CD103 + CD39 + ) Sorting the white blood cells into a flow collecting pipe, wherein the result is shown in fig. 2, fig. 2 is a flow chart showing a flow gate strategy, gates are drawn step by step in a two-dimensional scatter diagram form, and LD negative CD45 positive gates represent white blood cells in the direction shown by an arrow; CD19 negative CD3 positive represents T lymphocytes isolated from leukocytes; cyan: CD8 positive CD4 negative represents CD8T cells isolated from T lymphocytes; CD103 positive CD39 positive represents a tumor infiltration depleted T lymphocyte population isolated from CD8T lymphocytes.
S4, verifying the sorting efficiency, and carrying out differential gene expression analysis on the cell population of the sample injection group and the control group of the sorted tumor infiltration depleted T lymphocyte population by bulk transcriptome sequencing.
Based on gene expressionThe analysis showed that tumor infiltration depletes the T lymphocyte population (CD 3 + CD8 + CD103 + CD39 + ) Significantly expressed gene CD8A, ITGAE (encoding CD 103) gene, validating tumor infiltration depleted T lymphocyte population (CD 3) + CD8 + CD103 + CD39 + ) As a result, as shown in fig. 3, fig. 3 shows the results of differential gene expression analysis between the tumor-infiltration-depleted T lymphocytes and the control group, the left gene represents a gene highly expressed in the tumor-infiltration-depleted T lymphocytes and statistically different, and the right gene represents a gene highly expressed in the control group and statistically different, with low expression in the tumor-infiltration-depleted T lymphocytes.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While the obvious variations or modifications which are extended therefrom remain within the scope of the claims of this patent application.
Claims (7)
1. A method for sorting lung cancer tissue tumor infiltration depleted T lymphocytes by flow cytometry, comprising the steps of:
s1, preparing single-cell suspension, and digesting the treated lung cancer tumor tissue by collagenase to obtain single-cell suspension;
s2, adding an antibody into a single cell suspension for incubation, taking two equal single cell suspensions according to a TILs identification scheme, wherein the antibody CD45/CD3/CD19/CD4/CD8/CD39/CD103/L-D is added into one single cell suspension, and dyeing and incubating for 15min at room temperature, and taking the single cell suspension as a sample injection component of a follow-up flow cytometer to sort and exhaust T cells (LD - CD45 + CD3 + CD19 - CD8 + CD4 - CD103 + CD39 + ) The other single cell suspension was then stained with antibody CD45/CD3/CD19/CD8/CD4/CD127/CD25/L-D at room temperature for 15min, and used as a control group for a subsequent flow cytometer to sort non-depleted cell population tregs (LD - CD45 + CD3 + CD19 - CD8 - CD4 + CD127 - CD25 + ) Waiting for on-machine detection;
s3, flow-type sorting target cell groups, respectively loading sample injection group single cell suspension after incubation of antibodies and control group single cell suspension, analyzing by a flow cytometry, and carrying out tumor infiltration depletion on T lymphocytes (CD 8) + CD103 + CD39 + ) Setting a ring gate, adjusting the sorting angle of the flow cytometer, and sorting cells;
s4, verifying the sorting efficiency, and carrying out differential gene expression analysis on the cell population of the sample injection group and the control group of the sorted tumor infiltration depleted T lymphocyte population by bulk transcriptome sequencing.
2. The method for flow cytometry sorting lung cancer tissue tumor infiltration depleted T lymphocytes according to claim 1, wherein in step S1:
the treatment of lung cancer tumor tissue includes: after fresh tumor tissue is collected, the fresh tumor tissue is completely soaked in RPMI+10% FBS solution at the temperature of 4 ℃ and is placed on ice and stored in an ice box;
the collagenase digestion treatment includes: rinsing fresh tissue samples stored for no more than 2 hours with 1 XPBS at 4 ℃, cutting up the tissue samples with a sterilizing scalpel blade, transferring the tissue samples to a 2mL centrifuge tube filled with a tissue digestion solution, fully and uniformly mixing the tissue samples, carrying out water bath at 37 ℃ for 1 hour, filtering the tissue samples with a 16-gauge needle and a 40um cell filter screen after complete digestion, and carrying out resuspension after centrifugation to calculate the yield and the activity.
3. The method of sorting lung cancer tissue tumor infiltration depleted T lymphocytes of claim 2, wherein the tissue digestion solution employs complex media+0.225mg/mL collagenase type IV.
4. The method for selecting lung cancer tissue tumor infiltration and depletion of T lymphocytes according to claim 1, wherein in step S2, before the flow cytometer is started up, the suspension after incubation of the antibody is washed with PBS, put into a centrifuge, centrifuged at 500g centrifugal speed for 5min, resuspended with PBS, filtered through a 40 μm cell sieve and sterilized, and then used as a sample injection for a subsequent flow cytometer, waiting for the start-up.
5. The method for sorting lung cancer tissue tumor infiltration depleted T lymphocytes of claim 4, wherein the flow cytometer uses phosphate buffer as a sheath fluid and washes 1.5mL enzyme free centrifuge tubes with fetal calf serum, the enzyme free centrifuge tubes are placed on flow collection tubes in a sorting bin of the flow cytometer.
6. The method for sorting tumor-infiltrating depleted T lymphocytes in lung cancer tissue according to claim 1, wherein in step S3, the L-D/CD45 gated lymphocyte is used to obtain a tumor-infiltrating depleted T lymphocyte population (CD 3) sequentially by the gating strategy of CD19/CD3, CD8/CD4, CD103/CD39 + CD8 + CD103 + CD39 + ) And sorted into a flow collection tube.
7. The method for sorting lung cancer tissue tumor invasion-depleted T lymphocytes according to claim 1, wherein in step S4, the tumor invasion-depleted T lymphocyte population (CD 3 + CD8 + CD103 + CD39 + ) The gene CD8A and ITGAE (encoding CD 103) are obviously expressed, and the cell population in single cell suspension of the sample injection group is verified to be tumor infiltration depletion T lymphocyte population (CD 3) + CD8 + CD103 + CD39 + )。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310734868.3A CN116790500A (en) | 2023-06-20 | 2023-06-20 | Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310734868.3A CN116790500A (en) | 2023-06-20 | 2023-06-20 | Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790500A true CN116790500A (en) | 2023-09-22 |
Family
ID=88037070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310734868.3A Pending CN116790500A (en) | 2023-06-20 | 2023-06-20 | Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790500A (en) |
-
2023
- 2023-06-20 CN CN202310734868.3A patent/CN116790500A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3070865B2 (en) | Subset of human progenitor cells | |
| US5804442A (en) | Process for preparing macrophages, kits, and composition for the use of this process | |
| CN111440773B (en) | Optimized culture method for T cell in-vitro chimeric antigen receptor modification and amplification | |
| CN104877965B (en) | A kind of method for preparing mature erythrocyte | |
| CN109706180A (en) | A kind of umbilical cord mesenchymal stem cells, which are overexpressed IDO, enhances immunosuppressive method and application | |
| KR20030033006A (en) | Method of proliferating natural killer cells | |
| CN107400658A (en) | A kind of method that CD8+T cells are isolated and purified out from liver cancer tissue | |
| CN108486039B (en) | Method for inducing human adipose-derived stem cells to differentiate into testicular interstitial cells by using small molecules | |
| CN112852725B (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN117230012A (en) | Human colon cancer organoid culture method and application thereof | |
| CN116790500A (en) | Method for sorting lung cancer tissue tumor infiltration depleted T lymphocyte by flow cytometry | |
| CN112662631A (en) | CAR-T cell perfusion culture method | |
| Andrews et al. | The limited immunocompetence of thymocytes within murine thymic nurse cells | |
| CN110055219B (en) | Method for preparing heterogeneous hematopoietic stem and progenitor cells by using non-mobilized peripheral blood | |
| CN110548138B (en) | Aptamer modified alpha-Gal liposome and preparation method and application thereof | |
| CN108165530B (en) | Separated and purified mouse tumor tissue infiltration CD4+CD25+Method for regulatory T cells | |
| CN111172104A (en) | Separation culture method of umbilical blood mesenchymal stem cells | |
| CN102260647A (en) | Method for isolating and purifying breast cancer stem cells | |
| CN114149977A (en) | Method for immortalizing mouse lung microvascular endothelial cells to obtain extracellular vesicles | |
| CN113249326A (en) | Method for screening tumor antigen specificity TCR sequence | |
| CN108588021A (en) | The method of separation and purifying TIL, acquisition CD4+CD25+ cell subsets | |
| CN111500537A (en) | Method for promoting dendritic cell maturation and enhancing functions of dendritic cells by using fatty acid and application of method | |
| CN113046314B (en) | Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells | |
| CN113046315B (en) | Method for obtaining decidua-like natural killer cells by in vitro induction of peripheral blood natural killer cells | |
| CN115125251B (en) | Method for efficiently obtaining specific fully human monoclonal antibody gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |