Isolation and purification of human metaphase immune cells
Isolation and purification of human metaphase immune cells
The metaphase immune cell population is the biological basis of maternal-fetal immune tolerance, and its composition is extremely special, mainly composed of special types of NK cells (CD56 hrighCD16-) (～70%), T cells (～10%), and monocyte macrophages (～15%), which play a different immunoregulatory role locally at the maternal-fetal interface from that in the periphery through the expression of special activation markers and the production of a large number of cytokines, and also regulate the growth and migration of trophoblasts through paracrine effects, thus playing an important role in the maintenance of pregnancy. Through the expression of special activation markers and the production of a large number of cytokines, they play a different role from the periphery in the local maternal-fetal interface, forming the immunodominance of the Th2 subpopulation, and regulating trophoblast cell growth, differentiation, and migration through paracrine effects, thus playing an important local regulatory role in maintaining pregnancy.
Principle
The basic principle of isolation and purification of human meconium immune cells is that a large number of immune-related cells are gathered in the uterine meconium during early pregnancy (7-9 weeks). The meconium is taken out from the aborted tissues, digested with enzymes, ground and filtered, and centrifuged in a density gradient, and then the single nucleated cells in the density layer of 40%-60% are aspirated, and the meconium immune cells can be obtained through short-term cultivation. Then, according to the morphology, phenotype and functional characteristics of different cell groups, immunomagnetic bead sorting and flow cytometry sorting were applied to identify, isolate and purify the cells, which provided the basis for further analysis of the structure and function of each subgroup of cells.
Operation method
Isolation and purification of human metaphase immune cells
Principle
The basic principle of isolation and purification of human meconium immune cells is that a large number of immune-related cells are gathered in the uterine meconium during early pregnancy (7-9 weeks). The meconium is taken out from the aborted tissues, digested with enzymes, ground and filtered, and centrifuged in a density gradient, and then the single nucleated cells in the density layer of 40%-60% are aspirated, and the meconium immune cells can be obtained through short-term cultivation. Then, according to the morphology, phenotype and functional characteristics of different cell groups, immunomagnetic bead sorting and flow cytometry sorting were applied to identify, isolate and purify the cells, which provided the basis for further analysis of the structure and function of each subgroup of cells.
Materials and Instruments
Reagents: Move The basic steps for the isolation and purification of human metaphase immune cells can be divided into the following steps: Caveat 1. Remove meconium or chorionic tissue as aseptically as possible and place in a sterile bottle containing 100 IU/ml penicillin and 100 mg/L streptomycin in pre-cooled D-Hanks solution or 10% fetal bovine serum in complete culture solution. Ensure that the tissue is in the ice box for the shortest possible time to the laboratory. 2. Rinse the tissue in saline as much as possible, and if the resulting cell suspension is high in erythrocytes, it can be lysed with an erythrocyte lysing solution for 2 minutes. 3. Isolated mononuclear cells can be cultured in vitro in DMEM/F12 cell culture medium containing 10% FBS with the addition of IL-15 (10 ng/ml) and/or IL-2 (100 IU/ml) to maintain the growth of metaphase NK cells. Every 3 days, half of the medium is changed and the appropriate concentration of cytokines is reintroduced. Cells can be cultured continuously for 7 to 14 days. The metaphase NK cell phenotype should be CD56brighCD16-CD3CD3-CD3CD3 -. For more product details, please visit Aladdin Scientific website.
1 x PBS solution, D-Hanks solution, RBC lysate, DMEM/F12 culture medium, fetal bovine serum, collagenase type IV, DNAase type I, Percoll stock solution, antibiotics, MACS buffer.
Instruments:
Ophthalmic scissors, forceps, sieves of different sizes (100 mesh, 300 mesh), various types of petri dishes, syringe handles, plastic centrifuge tubes of different sizes, 1.5 ml EP tubes, pipettes, bench-top freezing centrifuge, magnetic poles and magnetic bead sorting columns, carbon dioxide incubator, water bath shaker, flow cytometer.
I. Preparation of reagents
(1) Preparation of RBC lysate: take 0.16 mol/L NH4Cl 90 ml (770.256 mg); 0.17 mol/L Tris (pH7.4) 10 ml (205.938 mg) dissolved in 100 ml of triple-distilled water and adjust the pH value to 7.2 with HCl. The sterile lysate should be filtered to remove bacteria and autoclaving is not recommended. After filtering the bacteria, store at 4 ℃.
(2) DMEM/F12 culture solution preparation: DMEM/F12 dry powder 15.6 g dissolved in distilled water, add NaHCO3 1.2 g mixing, fixed to 1000 ml, with 56 g / L NaHCO3 solution to adjust the pH value of 7.2 ~ 7.4, filtered under aseptic conditions to remove bacteria, stored in -20 ℃.
(3) D-Hanks liquid preparation: take NaCl 8.00 g, KCl 0.40 g, Na2HPO4-12 H20 0.12 g, KH2 HPO40.06 g, NaHCO3 0.35 g, anhydrous dextrose 1.00 g, dissolve in 1000 ml of triple-distilled water, adjust the pH 6.8-7.0, autoclave in bottles, and then cooled at 4 ℃. Preserve at 4 ℃ for use.
(4) Percoll solution preparation: Percoll mother liquor (Percoll original 9 ml, 10 x PBS 1 ml); 20% Percoll working solution (Percoll mother liquor 2 ml, 1 x PBS 8 ml); 40% Percoll working solution (Percoll mother liquor 4 ml, 1 x PBS 6 ml); 60% Percoll working solution (Percoll mother liquor 4 ml, 1 x PBS 6 ml). Percoll working solution (Percoll mother liquor 6 ml, 1 x PBS 4 ml). The characteristics of Percoll working solution are shown in Table 14-1.
(5) Preparation of MACS buffer: 1 x PBS (pH7.2), 0.5% BSA, 0.22 μm filter membrane for sterilization, and stored at 4 ℃.
Pretreatment of ecdysis cells
(1) Wash fresh ecdysis tissue with 1 x PBS three times to remove blood clots and blood stains, and cut the remaining tissue into pieces of about 1 mm3.
(2) Add 0.1% Collagenase IV (can be combined with 50 μg/ml DNase I to digest the tissue according to the laboratory conditions) and digest the tissue for 1 hour at 37℃ in a water bath with shaking, then terminate the digestion with DMEM/F12 cell culture medium containing 10% FBS. After grinding and filtering through a 100-mesh sieve, the cell suspension was centrifuged at 300 g for 10 min, and the supernatant was discarded.
(3) Resuspend the cell pellet in 3 ml of D-Hanks solution and carefully spread it on the discontinuous Percoll density gradient interface (60%, 40% and 20% gradients) and centrifuge at 1000 g for 20 minutes.
(4) Aspirate the cells between the 40% and 60% gradient layers (p = 1.056-1.077), wash once with 1xPBS, and resuspend in DMEM/F12 cell culture medium containing 10% FBS. The cells were further purified and isolated according to the experimental requirements.
Isolation and purification of metaphase NK cells
(1) The obtained cells were planted in culture flasks and incubated at 37 ℃ in a 5% CO2 incubator overnight, and then the liquid was changed to remove the wall-adherent metaphase stromal cells, metaphase glandular epithelial cells and metaphase macrophages, and then collect the suspended cells, which were the preliminary purified metaphase lymphocytes.
(2) Filter the first purification of membrane lymphocytes through 300 mesh filter, remove the cell clumps, centrifuge, and resuspend the cells by adding 40 μl of MACS buffer for every 10 ' cells.
(3) Add 10 μl of NK Biotin-labeled antibody cocktail per 107 cells, mix well and incubate at 4 ℃ for 10 minutes.
(4) Add 30 μl of MACS buffer.
(5) Add 20 μl NK microbeads to every 107 cells, mix well and incubate at 4 ℃ for 15 minutes.
(6) Centrifuge the cell suspension at 300 g for 10 minutes and resuspend the cells with 500 μl of MACS buffer.
(7) Set up a magnetic bead sorting column in the magnetic field, and wash the column with 3 ml of 1 x PBS buffer.
(8) The labeled cell suspension was loaded onto the column and washed three times with 3 ml of MACS buffer. After leaving the magnetic field, wash the column 3 times with 3 ml of MACS buffer, and the positive selected cells flowing out of the sorting column are NK cells.
(9) Take 2 x 10/100 μl of the obtained cells, label them with anti-CD3, anti-CD56 and anti-CD16 antibodies, and then analyze and identify the purity of CD56 brighCD3 NK cells by FACSAria. If higher purity of ecdysteroid NK cells is required, the subpopulation of CD56 brighCD3 NK cells can be further sorted by flow assay, and the purity of ecdysteroid NK cells can be >95%.
Separation and purification of metaphase CD4+T cells
(1) The obtained cells were planted in culture flasks, placed in a 37 ℃, 5% CO2 incubator for overnight culture, and then changed the liquid, to remove wall-adherent metaphase stromal cells, metaphase glandular epithelial cells and metaphase macrophages, and then collect the suspended cells, which is the purified metaphase lymphocytes.
(2) Remove cell clumps by filtration through 300 mesh filter, centrifuge, and resuspend the cells by adding 100 μl of MACS buffer for every 10' cells.
(3) Add 20 μl of anti-CD4-microbead to every 107 cells, mix well and incubate at 4℃ for 15 minutes.
(4) Add 10-20 times the total volume of labeled cells to pre-cooled MACS buffer and centrifuge at 300 g for 10 minutes.
(5) Resuspend the cells by adding 100 μl of MACS buffer for every 10 cells.
(6) Set up a magnetic bead sorting column in the magnetic field and wash the column with 3 ml of MACS buffer.
(7) Put the labeled cell suspension on the column, wash the column 3 times with 3 ml of MACS buffer, and leave the CD4+ T cells in the column for positive selection, add 5 ml of MACS buffer, and rinse out the cells in the column under pressure when the magnetic field is released.
(8) Repeat the sorting with a new sorting column to obtain a higher purity of sorted CD4+ T cells.
(9) Take 2 x 105/100 μl cells, incubate them with FITC-labeled human CD4 antibody for 25 minutes at room temperature, avoiding light, and wash them once with PBS; the purity of the cells identified by FACS was >95%.
V. Isolation and purification of metaphase dendritic cells
(1) The obtained cells were planted in culture flasks and cultured at 37 ℃ in a 5% CO2 incubator for 2 hours, and then the metaphase single nucleated cells without DSC and DEC were obtained.
(2) Suspend the cells by passing them through a 30 μm pore size filter to remove cell clusters, centrifuge at 300 g for 10 min, and resuspend the cells by adding 80 μl of MACS buffer for every 10' cells.
(3) Add 15 μl of Biotin-conjugated CD11c antibody per 10 cells, mix well and incubate at 4 ℃ for 10 minutes.
(4) Wash the cells once with 3 ml of MACS buffer.
(5) Add 12 μl microbead-conjugated anti-Biotin to every 10' cells, mix well and incubate at 4 ℃ for 15 minutes.
(6) Centrifuge the cells and resuspend the cells with 500 μl MACS buffer.
(7) Set up a magnetic bead sorting column in the magnetic field, and wash the column with 3 ml of 1xPBS buffer.
(8) The labeled cell suspension was loaded onto the column, and the column was washed three times with 3 ml of MACS buffer, and the positively selected cells that flowed out of the column were the ecdysteroidal DCs.
(9) The purity of the ecdysteroidal CD11c+DCs obtained was analyzed by FACSAria after taking 2 x 10/100 μl of obtained cells labeled with anti-CD11c surface antibody. If a higher purity of metaphase DC is required, the CD11c+DC subpopulation can be further sorted by flow-through.