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Isolation and in vivo Transfer of Antigen Presenting Cells
抗原递呈细胞的分离和体内转移

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Abstract

Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their antigen presenting properties.
As a percentage of total mononuclear cells, an average spleen contains approximately 1-3% myeloid dendritic cells (DCs). In absolute numbers, this translates to approximately 0.6-1.8 x 106 DCs. To enhance the number of DCs in spleen, mice were injected subcutaneously with cells from a cultured melanoma cell line (B16.Flt3L) which has been engineered to express the fms-related tyrosine kinase 3 ligand (Flt3L) (Mach et al., 2000). This protein is a growth factor homologous to colony stimulating factor-1 and plays a critical role in the differentiation of hematopoietic stem cells. Administration of this protein into mice as a purified protein results in the expansion of both CD8αPos and CD8αNeg DC subsets in multiple organs. Similar expansion is also seen in mice that have been implanted with tumor cells overexpressing this protein (Mach et al., 2000). In our experience, up to 60% of the total mononuclear cells in a spleen from a mouse with a palpable B16.Flt3L tumor can be CD11c positive dendritic cells, thereby giving a total yield of up to 5 x 107 DCs per mouse. A schematic illustrating the cell enrichment protocol is included in Figure 1, and representative data on purity of cell populations obtained with this protocol is shown in Figure 2.

Keywords: Isolation(隔离), Antigen presenting cell (APC)(抗原提呈细胞(APC)), Dendritic Cell(树突状细胞), Purification(净化), Antigen pulsing(Antigen脉冲)

Materials and Reagents

  1. Source of splenocytes: 6-8 week old female C57BL/6 mice (Jackson ImmunoResearch Laboratories)
  2. Murine B16.Flt3L melanoma cell line [as described by Mach et al. (2000)]
  3. Ultrapure water
  4. 0.05% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25300-054 )
  5. Isoflurane (Sigma-Aldrich, catalog number: CDS019936 -250MG)
  6. Collagenase D (Roche Diagnostics, catalog number: 11088858001 )
  7. DNase I, dry powder (QIAGEN, catalog number: 79254 )
  8. 70% Ethanol (prepared from 200 proof ethanol) (Thermo Fisher Scientific, catalog number: 9-6705-004-220 )
  9. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 )
  10. RPMI-1640 medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11875-119 )
  11. DMEM medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11995-073 )
  12. 200 mM L-glutamine (Life Technologies, catalog number: 25030081 )
  13. MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
  14. MEM essential amino acids (Life Technologies, catalog number: 11130-051 )
  15. β-mercaptoethanol (Life Technologies, InvitrogenTM, catalog number: 21985-023 )
  16. Sodium pyruvate (Life Technologies, catalog number: 11360-070 )
  17. HEPES (Life Technologies, InvitrogenTM, catalog number: 15630 )
  18. Phosphate buffered saline (PBS) (Ca2+ and Mg2+ free, pH 7.2) (Life Technologies, InvitrogenTM, catalog number: 20012-050 )
  19. Dulbecco’s PBS (DPBS) with Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14040-182 )
  20. 0.5 M Ethylenediaminetetraacetate (EDTA) solution (Life Technologies, catalog number: 15575-020 )
  21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  22. Fetal calf serum (Atlanta Biologicals, catalog number: S11050 )
  23. Penicillin/streptomycin (Life Technologies, InvitrogenTM, catalog number: 15140-163 )
  24. Trypan blue (dry powder) (Sigma-Aldrich, catalog number: T6146 -5G)
  25. Magnetic beads conjugated with anti-mouse CD19 (Miltenyi Biotech, catalog number: 120-000-323 )
  26. Magnetic beads conjugated with anti-mouse CD11c (Miltenyi Biotech, catalog number: 130-152-001 )
  27. CD8αPos mouse DC isolation kit (Miltenyi Biotech, catalog number: 130-091-169 )
  28. Fc-gamma receptor blocking antibody (Clone 2.4G2) (BD Biosciences, catalog number: 553141 ).
  29. Anti-mouse CD11c-FITC (BD Biosciences, catalog number: 553801 )
  30. Anti-mouse CD8α-PerCP (BD Biosciences, catalog number: 553036 )
  31. Anti-mouse B220-PE (BD Biosciences, catalog number: 553090 )
  32. 0.08% trypan blue (see Recipes)
  33. Serum free DMEM and RPMI media (see Recipes)
  34. Complete RPMI and DMEM media (see Recipes)
  35. MACS buffer (see Recipes)
  36. FACS staining buffer (see Recipes)
  37. 10x collagenase D solution (see Recipes)

Equipment

  1. 1 ml syringes (BD, catalog number: 26048 )
  2. 23 G1 needle (BD, catalog number: 305145 )
  3. 100 mm Petri dishes (Thermo Fisher Scientific, catalog number: 0875712 )
  4. Surgical instruments (Kent Scientific, catalog number: INSMOUSEKIT )
  5. Cell strainer (70 µm) (BD, catalog number: 352350 )
  6. Large Petri plates (Thermo Fisher Scientific, catalog number: FB0875712 )
  7. Vacuum filtration system (500 ml, 0.22 um) (Corning, catalog number: 431097 )
  8. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  9. Magnetic stand MACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
  10. Wide-bore 200 μl pipette tips (PerkinElmer, catalog number: 111623 )
  11. Corning ultra-low attachment 96-well plates (Corning, catalog number: CLS3474-24EA )

Procedure


Figure1. Scheme of cell enrichment protocol

  1. Implantation of B16.Flt3L melanoma in mice
    1. The B16.Flt3L melanoma cell line is grown to confluence in complete DMEM medium in standard cell culture flasks (25 cm2), and the cells are harvested by trypsinization. For this, aspirate the medium from the culture, and wash the adherent cells with sterile PBS. After washing, add 5 ml Trypsin EDTA (0.05%) solution and incubate at 37 °C for 5 min.
    2. After incubation, gently tap the flask on one side to lift the cells off of the surface of the vessel.
    3. Add 20 ml of complete DMEM medium to quench the protease digestion.
    4. Harvest cells from the flask by gently pipetting up and down three times, and then collecting the medium into a sterile 50 ml centrifuge tube.
    5. Pellet cells by centrifuging at 300 x g for 5 min at 4 °C. Wash the cells three times with PBS and resuspend in PBS to a density of 108 cells/ml.  
    6. Inject mice with 100 µl cell suspension (107 cells) subcutaneously into a single site in the area of the shoulder at the base of the neck.  This and all other procedures involving animals must be in accordance with and approved by the Institutional Animal Use and Care Committee (IACUC).
    7. Allow the tumor to grow until palpable or visible (2-10 mm in diameter, usually 7-10 days) before sacrificing animals for harvesting organs.

  2. Preparation of splenic single cell suspension
    1. Anesthetize mice with isoflurane. A typical approach for anesthesia involves placing the animal in an induction chamber connected to an oxygen source and isofluorane vaporizer, and adjusting oxygen flow to 0.9 liters/min and the isofluorane vaporizer to 3.5%. As soon as mice become unresponsive, they are sacrificed by cervical dislocation.
    2. Sterilize the left side of the abdomen by spraying the skin with 70% ethanol.
    3. Working under clean, aseptic conditions, make an incision under the rib cage on the left side of the abdomen and extract spleen using scissors.  Place in sterile serum free RPMI medium.
    4. From this step onwards, work in the biosafety cabinet and perform all the steps under sterile conditions.
    5. Wash the aseptically extracted spleen by submerging it in 5 ml of serum-free RPMI medium, and then transfer to a fresh 100 mm diameter Petri dishes. Note that because of the high yield of splenocytes from B6.Flt3L tumor bearing mice, one spleen is sufficient for most experiments, and this protocol is for the processing of a single spleen. For larger experiments involving pooling of two or more spleens, volumes may need to be scaled up proportionately.
    6. Cut spleen tissue into approximately 2 mm3 pieces using scissors and incubate in 10 ml of collagenase D (~200 units) and DNase I (10 μg/ml) solution at 37 °C for 30 min. Make sure the tissue pieces are completely immersed in the solution.
    7. Place a 70 µm cell strainer placed in a fresh Petri dish, and transfer the tissue pieces to it by pipetting with a 25 ml pipette.
    8. Crush the splenic pieces by gently compressing the tissue fragments against the mesh with the syringe plunger. Wash the mesh filter with 5 ml of complete RPMI medium to wash cells through the filter and into the collection dish. Use of 70 µm mesh filter removes larger cell aggregates and helps in generation of single cell suspension.
    9. Pellet cells by centrifuging at 300 x g for 5 min.
    10. Discard supernatant and add 2 ml RBC lysis buffer to the pellet.
    11. Mix gently by pipetting thrice with a 1 ml pipette and further incubate at room temperature for 5 min. Avoid longer incubations with RBC lysis buffer, since this may compromise cell viability.
    12. Neutralize the RBC lysis buffer by adding complete RPMI medium (12 ml per spleen) to the resulting suspension and centrifuge at 300 x g for 5 min.
    13. Resuspend the cell pellet in MACS buffer, using 0.5 ml per spleen. Filter through 70 µm cell strainer to remove cell clumps to prevent MACS column from clogging.

  3. Purification of CD8αPos DCs and CD8αNeg DCs from splenic single cell suspension
    Isolation of the CD8αPos DCs and CD8αNeg DCs from splenic cell suspension is a three-step protocol as illustrated in Figure 1. It relies on depletion of undesired populations like B, T and NK cells in the first step, followed by positive selection of CD8αPos DCs in the second step, and positive selection of CD8αNeg dendritic cells in the third step. Keep the cells on ice and use pre-cooled buffers to maintain 2-8 °C working temperature to prevent antibody capping and non-specific binding to irrelevant cells.
    1. Determine the cell number, and adjust concentration to approximately 5 x 108 cells/ml in MACS buffer containing 20 µg/ml Fc-gamma receptor blocking antibody (2.4G2).
    2. For each 108 total cells, add 100 µl of biotin antibody cocktail provided in the CD8αPos DC isolation kit. The antibody cocktail binds to B cells, T cells and NK cells.
    3. Mix thoroughly and incubate in ice cold water (4-8 °C) for 15 min.
    4. For each 108 total cells, add 150 µl of MACS buffer and 100 µl of anti-biotin beads provided in the CD8αPos DC isolation kit.
    5. Mix gently and incubate again in ice cold water (4-8 °C) for 15 min.  
    6. Add 12 ml MACS buffer and pellet cells by centrifuging at 300 x g for 5 min.
    7. Discard supernatant and resuspend the cell pellet in 2 ml of MACS buffer.
    8. Place a fresh MACS column in the magnetic field of an appropriate MACS separator.
    9. Equilibrate column by washing with 5 ml of MACS buffer.
    10. Pipette the cell suspension gently in the middle of the column.
    11. Collect unlabeled cells that flow through the column followed by washing with 10 ml MACS buffer.  The unlabeled flow through thus collected is enriched for DCs.
    12. Pellet the enriched DC fraction by centrifuging at 300 x g for 5 min.
    13. Resuspend the cells into 1 ml MACS buffer and add 200 µl of anti-CD8α conjugated Miltenyi magnetic bead suspension and incubate in ice cold water (4- 8 °C) for additional 15 min.
    14. Repeat steps C6-11.  The CD8αPos dendritic cells are bound with the anti-CD8α magnetic beads in this enrichment method, and are thus retained in the column.  The flow through of the column is enriched for CD8αNeg DCs and depleted for CD8αPos dendritic cells. The flow through should be retained to use for isolation of CD8αNeg dendritic cells in steps C18-22 below.   
    15. To elute the CD8αPos DCs from the column, gently remove the column from the magnetic field, add 5 ml MACS buffer and purge the column firmly with a syringe plunger.
    16. For enhanced purity, the CD8αPos enriched cells are passed through two consecutive columns. For this, pellet the eluted cells in step C15, resuspend with 2 ml MACS buffer and repeat steps 8-15.
    17. Pellet the collected CD8αPos DCs by centrifugation, resuspend in 0.2 ml medium and count. Keep the cells on ice until needed to enhance viability.  
    18. For isolation of CD8αNeg DCs, use the unlabeled cells collected as the column flow through in step C16. Pellet the cells by centrifugation and resuspend in 300 µl of MACS buffer.
    19. Add 100 µl of anti-CD11c magnetic beads and incubate in ice cold water (4- 8 °C) for 15 min.  
    20. Repeat steps C6-10.
    21. Wash column with 10 ml of MACS buffer. Elute the column retentate containing enriched CD8αNeg DCs.
    22. Pellet the cells and resuspend in 2 ml MACS buffer and pass them through a second MACS column to increase the purity if required.

  4. Isolation of B cells from splenic cell suspension
    1.  Determine the cell number.
    2. Add 200 µl MACS buffer containing 20 µg/ml Fc-gamma receptor blocking antibody (2.4G2), and 150 µl of anti-CD19 conjugated magnetic beads.
    3. Mix thoroughly and incubate in ice cold water (4-8 °C) for 10 min.
    4. Add 12 ml MACS buffer and pellet cells by centrifuging at 300 x g for 5 min.
    5. Discard supernatant and resuspend the cell pellet 2 ml of MACS buffer.
    6. Place a MACS column in the magnetic field of an appropriate MACS separator.
    7. Equilibrate column by washing with 5 ml of MACS buffer.
    8. Pipette the cell suspension gently in the middle of the column.
    9. This is a positive selection, so the target cells will be retained in the MACS column. To elute the cells from the column, gently remove column from magnetic field, add 5 ml MACS buffer and purge the column firmly with a plunger to collect CD8αPos DCs.

  5. Pulsing APCs with antigens and cell transfer
    1. Count live cells after MACS enrichment procedure by trypan blue exclusion.
    2. Plate cells in low-binding U-bottom 96 well plates at a density of 106 cells per well in 250 µl complete RPMI containing antigen of interest at desired concentration. Pulse the cells with antigen (typically at concentration of 100 nM, although this may vary depending on the particular antigen) for one hour in 37 °C incubator maintaining 5% CO2. Use of low binding plates allows for easier recovery of adherent cell populations.
    3. Harvest cells by pipetting up and down 5 times with the wide-bore tips. Pool cells from multiple wells and transfer to 15 ml tube. Add complete RPMI medium to 10 ml and pellet the cells by centrifuging at 300 x g for 5 min. Wash twice with PBS.
    4. Count the cells again and resuspend in PBS to a cell density of 107 cells/ml. Use these cells for injection into mice by the preferred route of administration.

Representative data


Figure 2. Pre- and post-enrichment analysis of splenic subsets. B and Dendritic cells are identified as B220 positive (R1, 38.5%) and CD11c positive (R2, 25.5%) live cells present in the spleens of B16.Flt3 melanoma bearing mice on day 8 after tumor implantation. In the second plot, gradient of CD8α expression can be observed on CD11c positive cells (R3 gate). In the post-enrichment plots, the relative purity of B cells, CD8αPos and CD8αNeg DCs is ascertained. The staining for CD11c in the enriched CD8αNeg DC subset competes with the anti-CD11c beads used to isolate these cells, thereby resulting in the observed gradient of CD11c-flourescence staining. In all cases, the purity of cells was found to be more than 95%.

Recipes

  1. 0.08% trypan blue
    Dissolve 0.08 g trypan blue in 100 ml ultrapure water
    Filter through 70 µm strainer to remove undissolved aggregates
  2. Serum free DMEM and RPMI media
    Mix all of the following ingredients in biosafety hood for either DMEM or RPMI media depending on your need
    Sterilize media by passing through 0.22 µm vacuum filtration system
    500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine
    5 ml MEM nonessential amino acids (100x, 10 mM)
    5 ml HEPES buffer (1 M)
    5 ml L-glutamine (200 mM)
    0.5 ml 2-mercaptoethanol (5.5 x 10-2 M)
  3. Complete RPMI and DMEM media
    Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media
  4. MACS buffer
    Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca2+ and Mg2+ free, pH 7.2)
    Filter sterile and degas the buffer by applying vacuum for at least 15 min prior to use to avoid air bubbles from blocking the column
  5. FACS staining buffer
    Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer
  6. 10x collagenase D and DNase 1 stock solution
    Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca2+ and Mg2+ to obtain a solution of approximately 1,000-2,000 Units of collagenase activity per ml
    This 10x stock solution that can be aliquoted as 1 ml vials and stored at -20 °C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use

Acknowledgments

This work was supported by NIH/NIAID grant AI45889 to S.A.P. Flow cytometry studies were carried out using FACS core facilities supported by the Einstein Cancer Center (NIH/NCI CA013330) and Center for AIDS Research (NIH/NIAID AI51519).

References

  1. Arora, P., Baena, A., Yu, K. O., Saini, N. K., Kharkwal, S. S., Goldberg, M. F., Kunnath-Velayudhan, S., Carreno, L. J., Venkataswamy, M. M., Kim, J., Lazar-Molnar, E., Lauvau, G., Chang, Y. T., Liu, Z., Bittman, R., Al-Shamkhani, A., Cox, L. R., Jervis, P. J., Veerapen, N., Besra, G. S. and Porcelli, S. A. (2014). A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens. Immunity 40(1): 105-116.
  2. Mach, N., Gillessen, S., Wilson, S. B., Sheehan, C., Mihm, M. and Dranoff, G. (2000). Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res 60(12): 3239-3246.

简介

体内转移抗原呈递细胞是免疫学家用来检查所选择的细胞群体的抗原呈递效力的方法。该方法最常用于分析蛋白质抗原对MHC I类或II类限制性T细胞的呈递,但其也可用于非常规抗原如CD1呈递脂质的研究。在最近关于CD1d限制性糖脂抗原呈递到天然杀伤T细胞的研究中,我们比较脾B细胞,CD8αpos 树突细胞(DC)和CD8α Neg sup> DC(Arora et al。,2014)。该方案描述了用于分离这些细胞群体和将其转移到受体小鼠中以分析其抗原呈递性质的详细方法。作为总单核细胞的百分比,平均脾脏含有约1-3%骨髓树突状细胞(DC)。在绝对数量上,这转换为大约0.6-1.8×10 6个DC。为了增强脾中DC的数目,向小鼠皮下注射来自经过工程改造以表达fms相关的酪氨酸激酶3配体(Flt3L)的培养的黑素瘤细胞系(B16.Flt3L)的细胞(Mach等人。,2000)。该蛋白是与集落刺激因子-1同源的生长因子,并在造血干细胞的分化中起关键作用。将这种蛋白质作为纯化的蛋白质施用到小鼠中导致多个器官中的CD8αpos pos和CD8α阳性DC亚群的扩增。在已经植入过表达这种蛋白质的肿瘤细胞的小鼠中也观察到类似的扩增(Mach等人,2000)。根据我们的经验,来自具有可触知的B16.Flt3L肿瘤的小鼠的脾脏中高达60%的总单核细胞可以是CD11c阳性树突细胞,从而得到高达5×10 7个细胞/sup>每只小鼠的DC。图1中包括细胞富集方案的示意图,并且用该方案获得的关于细胞群纯度的代表性数据显示在图2中。

关键字:隔离, 抗原提呈细胞(APC), 树突状细胞, 净化, Antigen脉冲

材料和试剂

  1. 脾细胞来源:6-8周龄的雌性C57BL/6小鼠(Jackson ImmunoResearch Laboratories)
  2. 鼠B16Flt3L黑素瘤细胞系[如Mach等人(2000)所述]
  3. 超纯水
  4. 0.05%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25300-054)
  5. 异氟烷(Sigma-Aldrich,目录号:CDS019936-250MG)
  6. 胶原酶D(Roche Diagnostics,目录号:11088858001)
  7. DNase I,干粉(QIAGEN,目录号:79254)
  8. 70%乙醇(由200标准乙醇制备)(Thermo Fisher Scientific,目录号:9-6705-004-220)
  9. RBC裂解缓冲液(Sigma-Aldrich,目录号:R7757)
  10. 具有L-谷氨酰胺的RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-119)
  11. 具有L-谷氨酰胺的DMEM培养基(Life Technologies,Gibco ,目录号:11995-073)
  12. 200mM L-谷氨酰胺(Life Technologies,目录号:25030081)
  13. MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140-050)
  14. MEM必需氨基酸(Life Technologies,目录号:11130-051)
  15. β-巯基乙醇(Life Technologies,Invitrogen TM ,目录号:21985-023)
  16. 丙酮酸钠(Life Technologies,目录号:11360-070)
  17. HEPES(Life Technologies,Invitrogen TM ,目录号:15630)
  18. 磷酸盐缓冲盐水(PBS)(Ca 2+和Mg 2+,游离,pH 7.2)(Life Technologies,Invitrogen TM,目录号: 20012-050)
  19. 具有Ca 2+和Mg 2+的Dulbecco's PBS(DPBS)(Life Technologies,Gibco ,目录号:14040-182)
  20. 0.5M乙二胺四乙酸(EDTA)溶液(Life Technologies,目录号:15575-020)
  21. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  22. 胎牛血清(Atlanta Biologicals,目录号:S11050)
  23. 青霉素/链霉素(Life Technologies,Invitrogen TM ,目录号:15140-163)
  24. 台盼蓝(干粉)(Sigma-Aldrich,目录号:T6146-5G)
  25. 与抗小鼠CD19缀合的磁珠(Miltenyi Biotech,目录号:120-000-323)
  26. 与抗小鼠CD11c(Miltenyi Biotech,目录号:130-152-001)缀合的磁珠
  27. CD8α Pos 小鼠DC分离试剂盒(Miltenyi Biotech,目录号:130-091-169)
  28. Fc-γ受体阻断抗体(克隆2.4G2)(BD Biosciences,目录号:553141)。
  29. 抗小鼠CD11c-FITC(BD Biosciences,目录号:553801)
  30. 抗小鼠CD8α-PerCP(BD Biosciences,目录号:553036)
  31. 抗小鼠B220-PE(BD Biosciences,目录号:553090)
  32. 0.08%台盼蓝(见配方)
  33. 无血清DMEM和RPMI培养基(参见配方)
  34. 完成RPMI和DMEM介质(请参阅配方)
  35. MACS缓冲区(参见配方)
  36. FACS染色缓冲液(参见配方)
  37. 10x胶原酶D溶液(见配方)

设备

  1. 1ml注射器(BD,目录号:26048)
  2. 23 G1针(BD,目录号:305145)
  3. 100mm培养皿(Thermo Fisher Scientific,目录号:0875712)
  4. 外科器械(Kent Scientific,目录号:INSMOUSEKIT)
  5. 细胞过滤器(70μm)(BD,目录号:352350)
  6. 大培养皿(Thermo Fisher Scientific,目录号:FB0875712)
  7. 真空过滤系统(500ml,0.22μm)(Corning,目录号:431097)
  8. LS柱(Miltenyi Biotec,目录号:130-042-401)
  9. 磁性台MACS分离器(Miltenyi Biotec,目录号:130-042-302)
  10. 大孔200μl移液管吸头(PerkinElmer,目录号:111623)
  11. Corning超低附着96孔板(Corning,目录号:CLS3474-24EA)

程序


图1。 细胞富集方案方案

  1. B16.Flt3L黑素瘤在小鼠中的植入
    1. 使B16.Flt3L黑素瘤细胞系在完全DMEM中生长至汇合   培养基的标准细胞培养瓶(25cm 2)中,并且细胞是 通过胰蛋白酶消化收获。 为此,从中抽吸培养基 培养,并用无菌PBS洗涤贴壁细胞。 洗涤后, 加入5ml胰蛋白酶EDTA(0.05%)溶液并在37℃下孵育5分钟。
    2. 孵育后,轻轻敲打一边的烧瓶,以将细胞从容器的表面提起。
    3. 加入20ml完全DMEM培养基以淬灭蛋白酶消化
    4. 从烧瓶中收集细胞,轻轻地吸取上下三个 次,然后将培养基收集到无菌的50ml离心机中 管。
    5. 通过在4℃下以300×g离心5分钟沉淀沉淀细胞 C。 用PBS洗涤细胞三次,并重悬在PBS中 密度为10 8个细胞/ml。  
    6. 注射100微升细胞的小鼠 悬浮液(10μL细胞)皮下注射到区域中的单个位点 肩膀在颈部的底部。 这个和所有其他程序 涉及动物必须按照并经批准 机构动物使用和护理委员会(IACUC)。
    7. 允许   肿瘤生长直到可触及或可见(直径2-10mm,通常 7-10天),然后处死动物收获器官

  2. 脾单细胞悬液的制备
    1. 用异氟烷麻醉小鼠。 麻醉的典型方法 包括将动物放置在连接到的诱导室中 氧源和异氟烷蒸发器,并调节氧气流量 0.9升/分钟,异氟烷蒸发器为3.5%。 一旦小鼠 变得无反应,它们被颈椎脱臼所牺牲
    2. 用70%乙醇喷洒皮肤,消毒腹部左侧。
    3. 在清洁,无菌条件下工作,在下切口 肋骨左侧的腹部和提取脾使用 剪刀。 置于无菌无血清RPMI培养基中
    4. 从这一步开始,在生物安全柜中工作,并在无菌条件下执行所有步骤。
    5. 通过将无菌提取的脾浸没在5毫升中洗涤 无血清RPMI培养基,然后转移至新鲜的100mm直径 培养皿。 注意,因为脾细胞的高产率 B6.Flt3L肿瘤携带小鼠,一只脾对大多数是足够的 实验,这个协议是用于处理单个脾脏。   对于涉及两个或更多个脾的汇集的较大实验, 卷可能需要按比例增大。
    6. 切脾 使用剪刀将组织切成大约2mm的小块并在10℃下孵育   ml胶原酶D(〜200单位)和DNA酶I(10μg/ml)溶液 ℃30分钟。 确保组织片完全浸没 解决方案。
    7. 放置在新鲜的70微米细胞过滤器 培养皿,并通过用25分钟移液将组织片转移到它 ml移液管。
    8. 通过轻轻压缩脾碎片   组织碎片靠着注射器柱塞的网。 洗涤 滤网用5ml完全RPMI培养基通过洗涤细胞 过滤器和进入收集盘。 使用70μm网过滤器 更大的细胞聚集体,并有助于产生单细胞 暂停。
    9. 通过在300×g离心5分钟使沉淀细胞沉淀
    10. 弃去上清液,向沉淀中加入2ml RBC裂解缓冲液
    11. 通过用1ml移液管吸取三次轻轻混合,并进一步 在室温下孵育5分钟。 避免更长时间的孵化 RBC裂解缓冲液,因为这可能损害细胞活力
    12. 通过加入完全RPMI培养基(12ml)中和RBC裂解缓冲液 每脾)至所得悬浮液并在300×g离心5分钟 min。
    13. 重悬细胞沉淀在MACS缓冲液,使用0.5毫升 每脾。通过70μm细胞过滤器过滤以除去细胞团 防止MACS列阻塞。

  3. 纯化来自脾单细胞悬浮液的CD8α肿瘤细胞和CD8α肿瘤细胞 来自脾细胞悬浮液的CD8α支原体DCs和CD8α支原体DC的分离是如图1所示的三步骤方案。其依赖于不想要的群体如B, T和NK细胞,然后在第二步中阳性选择CD8α阳性DC,在第三步阳性选择CD8α阴性树突状细胞。保持细胞在冰上,并使用预冷缓冲液,以保持2-8°C的工作温度,以防止抗体封顶和非特异性绑定到不相关的细胞。
    1. 确定细胞数量,并将浓度调节至约5 x   10包含20μg/ml Fc-γ受体的MACS缓冲液中的10 8细胞/ml 阻断抗体(2.4G2)
    2. 对于每10个总细胞,加入100μl   的CD8α Pos DC分离试剂盒中提供的生物素抗体混合物。 抗体混合物结合B细胞,T细胞和NK细胞
    3. 充分混合并在冰冷的水(4-8℃)中孵育15分钟。
    4. 对于每个10 8个总细胞,加入150μl的MACS缓冲液和100μl 抗生物素珠提供在CD8αpos DC分离试剂盒中。
    5. 轻轻混合并再次在冰冷的水(4-8℃)中孵育15分钟。  
    6. 通过在300×g离心5分钟加入12ml MACS缓冲液和沉淀细胞。
    7. 弃去上清液并将细胞沉淀重悬在2ml MACS缓冲液中
    8. 将新鲜的MACS柱置于适当的MACS分离器的磁场中。
    9. 通过用5ml MACS缓冲液洗涤平衡柱。
    10. 在柱中间轻轻吸取细胞悬液。
    11. 收集未标记的流过柱子的细胞 用10ml MACS缓冲液洗涤。 因此,未标记的流量 收集富集DC。
    12. 通过在300×g离心5分钟来沉淀富集的DC部分。
    13. 将细胞重悬在1ml MACS缓冲液中,加入200μl抗CD8α 共轭的Miltenyi磁珠悬浮液并在冰冷下孵育 水(4-8℃)再搅拌15分钟
    14. 重复步骤C6-11。 CD8α支持树突状细胞与抗CD8α磁珠结合 在此富集方法中,因此保留在列中。 的 柱的流过物富集了CD8α Neg DCs并耗尽 CD8αpos 树突状细胞。 流通应保留用于 在下面的步骤C18-22中分离CD8α Neg 树突细胞。  
    15. 要从柱中洗脱CD8α Pos DC,请轻轻地从柱中取出柱   磁场,加入5毫升MACS缓冲液,并牢固地吹扫色谱柱 用注射器柱塞
    16. 为了提高纯度,将富集CD8α的Pos细胞通过两个连续的柱。 为了这, 在步骤C15中沉淀洗脱的细胞,用2ml MACS缓冲液重悬   重复步骤8-15。
    17. 将收集的CD8α Pos DCs沉淀 离心,重悬于0.2ml培养基中并计数。 保持细胞 冰直到需要增强活力。  
    18. 用于隔离 CD8α Neg DC,使用未标记的细胞作为柱流收集 通过。 通过离心沉淀细胞并重悬   300μlMACS缓冲液。
    19. 加入100μl的抗CD11c磁珠,并在冰冷的水(4-8℃)中孵育15分钟。  
    20. 重复步骤C6-10。
    21. 用10 ml MACS缓冲液洗柱。 洗脱含有富集的CD8α Neg DC的柱滞留物
    22. 沉淀细胞并重悬于2ml MACS缓冲液中并通过它们 通过第二MACS柱以提高纯度(如果需要)。

  4. 从脾细胞悬液中分离B细胞
    1.  确定单元格号。
    2. 加入200μlMACS缓冲液 20μg/ml Fc-γ受体阻断抗体(2.4G2)和150μl 抗CD19共轭磁珠
    3. 充分混合并在冰冷的水(4-8℃)中孵育10分钟。
    4. 通过在300×g离心5分钟加入12ml MACS缓冲液和沉淀细胞。
    5. 弃去上清液并重悬细胞沉淀2ml MACS缓冲液
    6. 将MACS列放置在适当的MACS分隔符的磁场中。
    7. 通过用5ml MACS缓冲液洗涤平衡柱。
    8. 在柱中间轻轻吸取细胞悬液。
    9. 这是一个积极的选择,所以目标单元格将被保留 MACS列。 要从柱中洗脱细胞,轻轻取出 柱从磁场中,加入5 ml MACS缓冲液,并吹扫色谱柱 牢固地用柱塞收集CD8αPosDC。

  5. 脉冲APCs与抗原和细胞转移
    1. 通过台盼蓝排除法计算MACS富集程序后的活细胞。
    2. 平板细胞在低结合的U-底96孔板中,以每孔10 6个细胞的密度在250μl含有目的抗原的完全RPMI中 。 用抗原脉冲细胞(通常在 浓度为100nM,尽管这可能根据不同而不同 特定抗原)在37℃培养箱中保持5%CO 2下培养1小时。 使用低结合板允许更容易回收粘附细胞 人口
    3. 通过吸移上下5次收获细胞 与大口径的提示。 池细胞从多个井和转移到 15 ml管。 添加完全RPMI培养基到10毫升和沉淀的细胞 在300×g离心5分钟。 用PBS洗涤两次。
    4. 计数 再次将细胞重悬于PBS中至10 7个细胞/ml的细胞密度。 使用这些细胞注射到小鼠的首选途径 行政。

代表数据


图2.脾脏亚群的前和后富集分析 B和树突细胞被鉴定为存在于细胞中的B220阳性(R1,38.5%)和CD11c阳性(R2,25.5%)活细胞肿瘤移植后第8天的B16.Flt3黑色素瘤携带小鼠的脾脏。在第二张图中,可以在CD11c阳性细胞(R3门)上观察到CD8α表达的梯度。在后富集图中,确定了B细胞,CD8αpos pos和CD8α阳性DC的相对纯度。富集的CD8α阴性DC亚群中CD11c的染色与用于分离这些细胞的抗CD11c珠竞争,从而导致观察到的CD11c-荧光染色的梯度。在所有情况下,发现细胞的纯度大于95%。

食谱

  1. 0.08%台盼蓝
    将0.08克台盼蓝溶解在100毫升超纯水中
    通过70μm过滤器过滤以除去未溶解的聚集体
  2. 无血清DMEM和RPMI培养基
    根据您的需要,将所有以下成分混合在生物安全罩中用于DMEM或RPMI培养基 通过0.22μm真空过滤系统对介质进行灭菌 500ml含L-谷氨酰胺的DMEM或500ml含L-谷氨酰胺的RPMI-1640 5ml MEM非必需氨基酸(100x,10mM) 5ml HEPES缓冲液(1M) 5ml L-谷氨酰胺(200mM) 0.5ml 2-巯基乙醇(5.5×10 -2 M)
  3. 完成RPMI和DMEM媒体
    加入50毫升热灭活的胎牛血清到无血清RPMI或DMEM培养基中,得到完全培养基
  4. MACS缓冲区
    将2ml的0.5M EDTA和10ml热灭活的胎牛血清加入到500ml磷酸盐缓冲盐水(PBS,Ca 2+和Mg 2+)中, pH 7.2)
    过滤灭菌,并在使用前通过使用真空至少15分钟使缓冲液脱气,以避免气泡堵塞柱子
  5. FACS染色缓冲液
    将叠氮化钠溶于MACS缓冲液中的0.05%(0.25g/500ml),得到FACS染色缓冲液
  6. 10x胶原酶D和DNase 1储备液
    在含有Ca 2+和Mg 2+的20ml PBS中溶解1g胶原酶D和0.2ml DNA酶1储液(1mg/ml,100x),得到每毫升大约1,000-2,000单位胶原酶活性的溶液
    该10x储备溶液可以等分为1ml小瓶并在-20℃下储存数周。在使用前立即用9ml无血清RPMI稀释1ml。

致谢

这项工作是由NIH/NIAID授予AI45889支持S.A.P.使用由爱因斯坦癌症中心(NIH/NCI CA013330)和AIDS研究中心(NIH/NIAID AI51519)支持的FACS核心设施进行流式细胞术研究。

参考文献

  1. Arora,P.,Baena,A.,Yu,KO,Saini,NK,Kharkwal,SS,Goldberg,MF,Kunnath-Velayudhan,S.,Carreno,LJ,Venkataswamy,MM,Kim,J.,Lazar-Molnar, E.,Lauvau,G.,Chang,YT,Liu,Z.,Bittman,R.,Al-Shamkhani,A.,Cox,LR,Jervis,PJ,Veerapen,N.,Besra,GSand Porcelli, 2014)。 树突状细胞的单个子集控制天然杀伤T细胞对多种糖脂抗原的反应的细胞因子偏好。 免疫力 40(1):105-116。
  2. Mach,N.,Gillessen,S.,Wilson,S.B.,Sheehan,C.,Mihm,M。和Dranoff,G。(2000)。 体内刺激的树突状细胞中的差异通过工程化分泌粒细胞 - 巨噬细胞集落刺激因子或Flt3-配体。 Cancer Res 60(12):3239-3246。
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引用:Arora, P., Kharkwal, S. S. and Porcelli, S. A. (2014). Isolation and in vivo Transfer of Antigen Presenting Cells. Bio-protocol 4(19): e1260. DOI: 10.21769/BioProtoc.1260.
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