参见作者原研究论文

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May 2015
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In vitro Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells
基于鼠源淋巴祖细胞的体外分化获得先天免疫性淋巴细胞的方法   

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Abstract

Subtypes of innate lymphoid cells (ILC), defined based on their cytokine secretion profiles and transcription factor expression, are important for host protection from pathogens and maintaining tissue homeostasis. ILCs develop from common lymphoid progenitors (CLP) in the bone marrow. Using the methods described here, we have previously shown that loss of the transcriptional regulator TOX (Thymocyte-selection associated HMG-box protein) leads to specific changes in ILC development and differentiation. Here, we describe how to obtain ILCs from in vivo isolated CLP grown in vitro.

Materials and Reagents

  1. 5 ml Syringe (BD biosciences, catalog number: 309646 )
  2. 25 G x 5/8 Needle (BD biosciences, catalog number: 305122 )
  3. 15 ml Conical Tubes (Corning, Falcon, catalog number: 352097 )
  4. 50 ml Conical Tubes (USA Scientific, catalog number: 1500-1811 )
  5. 60 x 15 mm culture dish (Corning, Falcon, catalog number: 351007 )
  6. Sterile 48 well plates, tissue culture treated (Greiner Bio-One GmbH, catalog number: 677180 )
  7. 5 ml and 10 ml serological pipets
  8. OP9-DL1 cells (not commercially available, must privately request from Dr. J. C. Zuniga-Pflucker, University of Toronto, Sunnybrook Research Institute)
  9. OP9-DL1 stromal cells (OP9-DL1 made by and obtained from Dr. J. C. Zuniga-Pflucker)
  10. Antibodies (all purchased from eBiosciences, Affymetrix)
    1. Blocking antibody Anti-CD16/CD32 Functional Grade Purified (clone 93) (eBioscience, Affymetrix, catalog number: 16-0161 )
    2. Anti-mouse lineage antibodies (concentrations as per vendor instructions), Fluorochromes were conjugated to FITC or APC (Lineage markers), APC-Cy7 (IL7Rα), PerCP eFluor® 710 (Flt-3) and PE-Cy7 (Ly6A/E)
      CD4 (clone 6K1.5)
      CD8α (53-6.7)
      CD3ε (145-2C11)
      CD11b (M1/70)
      CD11c (N418)
      CD19 (1D3)
      B220 (RA3-6B2)
      Gr-1 (RB6-8C5)
      Nk1.1 (PK136)
      Ter-119 (TER-119)
      Thy1.2 (30-H12)
    3. Common lymphoid progenitor defining cell surface antibodies (concentrations as per vendor instructions)
      Flt3 (A2F10)
      IL7Rα (A7R34)
      Ly6A/E (Sca-1) (D7)
  11. Dulbecco’s Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  12. Dulbecco’s Modified Eagle Medium, High Glucose (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  13. Minimum Essential Media (MEM) alpha, No Nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 12561-049 )
  14. Fetal Bovine Serum (FBS) (Omega Scientific, catalog number: FB-01 )
  15. 100x Penicillin-Streptomycin (PSA) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378-016 )
  16. Sodium Azide (Sigma-Aldrich, catalog number: S2002 )
  17. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  18. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  19. MEM nonessential amino acids (Mediatech, Corning® glutagroTM, catalog number: 25-025-CI )
  20. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630 )
  21. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054 )
  22. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A8022 )
  23. Recombinant mouse Interleukin 7 (IL-7) carrier free (Biolegend, catalog number: 577802 )
  24. Recombinant mouse Stem Cell Factor (SCF) carrier free (Biolegend, catalog number: 579702 )
  25. Recombinant mouse IL-33 carrier free (Biolegend, catalog number: 580504 )
  26. Mitomycin C (Santa Cruz Biotechnology, catalog number: sc-3514 )
  27. Phosphate buffered saline (PBS)
  28. NaN3
  29. FACS Buffer (see Recipes)
  30. Sterile Sorting Buffer (see Recipes)
  31. Innate lymphoid cell media (ILC media) (see Recipes)
  32. OP9-DL1 media (see Recipes)

Equipment

  1. Sterile Biosafety cabinet
  2. Incubator set at 37 °C, 5% CO2
  3. Vortex Genie 2 (Thermo Fisher Scientific, catalog number: 12-812 )
  4. Sterile FACS Tubes
  5. Cell sorter (BD Biosciences, model: FACS aria III )
  6. Reichert Bright-Line Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  7. Dissection scissors (Roboz Surgical Instrument Co., catalog number: RS-5912 )
  8. Forceps (Roboz Surgical Instrument Co., catalog number: RS-5137 )

Procedure

Part I. Maintaining OP9-DL1 cells

  1. Thawing OP9-DL1
    1. Prepare OP9-DL1 media (recipes) and place 10 ml media in a 15 ml conical tube.
    2. Thaw a prepared cyro-vial from liquid nitrogen (frozen in FBS plus 10% DMSO, 1ml total volume) by placing the tube in a 37 °C water bath until the ice is melted (approximately 3 min).
    3. Add this to 10 ml of OP9-DL1 media using a 1 ml micropipette and centrifuge 235 rcf for 5 min at room temperature.
    4. Carefully remove the media, leaving only the cell pellet.
    5. Add 10 ml fresh OP9-DL1 media to the tube and pipet up and down to dislodge the pellet.
    6. Add this 10 ml to a T75 flask containing 5 ml OP9-DL1 media for a total of 15B ml.
    7. Incubate at 37 °C, 5% CO2, split cells 1:3 every 3-4 day.

  2. Passaging OP9-DL1
    Note: OP9-DL1 cells will need to be split 1:3 every 3-4 d depending on growth kinetics (70%-75% confluence or approximately 0.6-1 x 106 cells in a T75 flask). If OP9-DL1 reaches a high density, they must be discarded, as they will turn into adipocytes.
    1. Prepare OP9-DL1 media (recipes) and place 10 ml media in a 15 ml conical tube.
    2. Remove media from T75 flask and add 7 ml Trypsin.
    3. Incubate 37 °C, 5% CO2 for approximately 3 min or until OP9-DL1 start to detach.
    4. Carefully remove Trypsin and immediately add 10 ml OP9-DL1 media.
    5. Wash walls of the flask with 10 ml pipet to dislodge OP9-DL1 cells.
    6. Centrifuge 260 x g for 5 min at 25 °C.
    7. Remove supernatant and add 1 ml OP9-DL1 media to pellet.
    8. Count cells using a hemocytometer and split appropriately (1 T75 flask should have approximately 300,000 cells).

  3. Plating OP9-DL1 for differentiation of CLP
    1. Prepare OP9-DL1 media (recipes) and place 10 ml media in a 15 ml conical tube.
    2. Remove media from T75 flask and add 7 ml Trypsin.
    3. Incubate 37 °C, 5% CO2 for approximately 3 min or until OP9-DL1 start to detach.
    4. Carefully remove Trypsin and immediately add 10 ml OP9-DL1 media.
    5. Wash walls of flask with 10 ml pipet to dislodge OP9-DL1 cells.
    6. Centrifuge 260 x g for 5 min at 25 °C.
    7. Remove supernatant and add 1 ml OP9-DL1 media to pellet.
    8. Count cells using a hemocytometer and plate OP9-DL1 at a density of 30,000 cells/well in 200-300 μl in a 48 well plate.
    9. Allow at least 6 h for cells to attach to surface.
    10. Add 1x Mitomycin-C (200-300 μl/well at a 50 µg/ml final concentration) to each well and incubate 37 °C, 5% CO2 for 25 min.
    11. Gently add 1 ml per well of warmed OP9-DL1 media to each well to wash out Mitomycin-C.
    12. Aspirate media and repeat step C11, 3 times.
    13. The OP9-DL1 feeder layer is now ready to plate isolated CLPs.

Part II. Differentiation of CLP to ILCs

  1. Common Lymphoid Progenitor (CLP) isolation and immunocytochemistry
    1. Prepare a 60 x 15 mm culture dish containing 3 ml FACS buffer (FACS buffer kept at 4 °C).
    2. Prepare a 5 ml Syringe with a 25G x 5/8 needle containing 5 ml PBS.
    3. Remove the mouse hind limb femur and tibia by cutting proximal to the joints as to have both ends of the bone open. Remove the femur completely with both ends cut.
    4. Holding the bone with a forceps, flush the Bone Marrow (BM) out of the bone by slowly adding the 5 ml PBS in the Syringe.
    5. Dissociate the BM by aspirating with the 5 ml Syringe/25G needle 2x.
    6. Add cells (8 ml) to a 15 ml conical tube.
    7. Centrifuge the cells for 5 min at 260 x g, 25 °C.
    8. Resuspend in 10 ml sterile sort buffer at 4 °C and count the cells by using a hemocytometer. Concentration of cells depends on manufacturer instructions for antibody staining. Here, a test was defined as 1 x 106 cells/test and was performed in a volume of 200 μl/test.
    9. Block by pre-incubating cells (1 x 106 cells/test) with CD16/CD32 on ice for 10 min.
    10. Stain for cell surface molecules using the specified antibodies listed under “antibodies” as per manufacturer instruction.
    11. Incubate at 4 °C for 30 min.
    12. Wash cells by adding 3 ml sterile sorting buffer and centrifuge at 235 x g (rcf) for 5 min.
    13. Resuspend in appropriate amount of sterile FACS buffer (see manufacturer instructions for cell numbers per test) and proceed to sorting.
    14. Gate and sort CLP as lineage negative, CD127+ Flt3+ Sca-1int (see Figure 1 for gating example) on an ARIA III or ARIA II using a 70 µm nozzle. Sort CLPs directly into a 1.5 ml eppendorf tube containing 300 μl ILC media supplemented with 20 ng/ml IL-7.
    15. Following sort, plate 7,500 CLPs/well in a 48 well plate in 300 μl cDMEM/IL-7 per well (sort should net approximately 15,000 CLPs/animal) on mitomycin-c treated OP9-DL1 (see part I, section C above).
    16. Feed with 300 μl fresh cDMEM media supplemented with 20 ng/ml IL-7 and 20 ng/ml SCF every three days. Replate on fresh OP9-DL1 at a concentration of 7,500 cells/well every six days. Add cytokines according to what ILC subtype is under investigation (i.e., for ILC-2 add 20 ng/ml IL-33).
    17. Cells can be analyzed for ILC using previously reported cell surface markers and transcription factors (see reference) as early as six days following plate-down of CLPs. If one wishes to polarize populations specifically to an ILC2 fate, IL-33 is added to the media at a concentration of 20 ng/ml (i.e., CLPs are sorted directly into media containing cytokine) (Figure 1).


      Figure 1. Gating strategy for the isolation of common lymphoid progenitor cells (CLPs) from bone marrow of wild-type mice. Numbers adjacent to outlined areas indicate percent cells in each gate. CLPs are specifically defined as Lin-CD127+Flt3+Sca-1+. Data are representative of 10 experiments with cells pooled from two mice in each.

Recipes

  1. FACS buffer (for 500 ml)
    480 ml phosphate buffered saline (PBS)
    15 ml FBS (3% final)
    5 ml of a 10% sodium azide solution (10 g NaN3 in 100 ml deionized water)
    Stored at 4 °C
  2. Sterile sorting buffer (for 500 ml)
    2.5 g BSA (0.5% final)
    12.5 HEPES (25 mM final)
    485 ml PBS
  3. ILC media
    DMEM high glucose
    10% FBS
    50 µM 2-mercaptoethanol
    1% penicillin-streptomycin
    1 mM sodium pyruvate
    1x non-essential amino acids
    20 mM HEPES (pH 7.4)
    IL-7 (20 ng/ml)
    SCF (50 ng/ml)
  4. OP9-DL1 media
    Alpha-MEM
    20% FBS
    1% penicillin-streptomycin

Acknowledgments

This work was supported by the U. S National Institutes of Health. This protocol was modified from reference (Seehus et al., 2015).

References

  1. Seehus, C. R., Aliahmad, P., de la Torre, B., Iliev, I. D., Spurka, L., Funari, V. A. and Kaye, J. (2015). The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat Immunol 16(6): 599-608.
  2. Wong, S. H., Walker, J. A., Jolin, H. E., Drynan, L. F., Hams, E., Camelo, A., Barlow, J. L., Neill, D. R., Panova, V., Koch, U., Radtke, F., Hardman, C. S., Hwang, Y. Y., Fallon, P. G. and McKenzie, A. N. (2012). Transcription factor RORalpha is critical for nuocyte development. Nat Immunol 13(3): 229-236.

简介

先天淋巴细胞(ILC)的亚型,基于其细胞因子分泌谱和转录因子表达来定义,对于宿主保护免于病原体和维持组织内稳态是重要的。 ILC从骨髓中的普通淋巴祖细胞(CLP)发展而来。 使用本文所述的方法,我们以前显示转录调节器TOX(胸腺细胞选择相关的HMG框蛋白)的损失导致ILC发展和分化的具体变化。 在这里,我们描述了如何从体内分离的CLP获得ILC。

材料和试剂

  1. 5ml注射器(BD biosciences,目录号:309646)
  2. 25 G x 5/8针(BD biosciences,目录号:305122)
  3. 15ml锥形管(Corning,Falcon,目录号:352097)
  4. 50ml锥形管(USA Scientific,目录号:1500-1811)
  5. 60×15mm培养皿(Corning,Falcon,目录号:351007)
  6. 无菌48孔板,组织培养处理(Greiner Bio-One GmbH,目录号:677180)
  7. 5ml和10ml血清移液管
  8. OP9-DL1细胞(不可商购,必须私人请求来自J.C.Zuniga-Pflucker,University of Toronto,Sunnybrook Research Institute)
  9. OP9-DL1基质细胞(由Dr.J.Zuniga-Pflucker制造并获得的OP9-DL1)
  10. 抗体(均购自eBiosciences,Affymetrix)
    1. 封闭抗体抗CD16/CD32功能级纯化(克隆93)(eBioscience,Affymetrix,目录号:16-0161)
    2. 抗小鼠谱系抗体(根据供应商的浓度 说明书),荧光染料与FITC或APC(Lineage 标记),APC-Cy7(IL7Rα),PerCP eFluor 710(Flt-3)和PE-Cy7 (Ly6A/E)
      CD4(克隆6K1.5)
      CD8α(53-6.7)
      CD3ε(145-2C11)
      CD11b(M1/70)
      CD11c(N418)
      CD19(1D3)
      B220(RA3-6B2)
      Gr-1(RB6-8C5)
      Nk1.1(PK136)
      Ter-119(TER-119)
      (30-H12)
    3. 限定细胞表面抗体的常见淋巴祖细胞(根据供应商说明的浓度)
      Flt3(A2F10)
      IL7Rα(A7R34)
      Ly6A/E(Sca-1)(D7)
  11. Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM ,目录号:14190-144)
  12. Dulbecco改良的Eagle培养基,高葡萄糖(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
  13. 最小必需培养基(MEM)α,无核苷(Thermo Fisher Scientific,Gibco TM ,目录号:12561-049)
  14. 胎牛血清(FBS)(Omega Scientific,目录号:FB-01)
  15. 100x青霉素 - 链霉素(PSA)(Thermo Fisher Scientific,Gibco TM ,目录号:10378-016)
  16. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  17. 2-巯基乙醇(Sigma-Aldrich,目录号:M7522)
  18. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM ,目录号:11360-070)
  19. MEM非必需氨基酸(Mediatech,Corning glutagro TM ,目录号:25-025-CI)
  20. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630)
  21. 胰蛋白酶-EDTA(0.05%),酚红(Thermo Fisher Scientific,Gibco TM,目录号:25300-054)
  22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A8022)
  23. 重组小鼠白细胞介素7(IL-7)载体(Biolegend,目录号:577802)
  24. 重组小鼠干细胞因子(SCF)载体(Biolegend,目录号:579702)
  25. 重组小鼠IL-33载体(Biolegend,目录号:580504)
  26. 丝裂霉素C(Santa Cruz Biotechnology,目录号:sc-3514)
  27. 磷酸盐缓冲盐水(PBS)
  28. NaN 3
  29. FACS缓冲区(参见配方)
  30. 无菌分选缓冲液(参见配方)
  31. 先天淋巴细胞培养基(ILC培养基)(参见配方)
  32. OP9-DL1介质(参见配方)

设备

  1. 无菌生物安全柜
  2. 孵育器设置在37℃,5%CO 2/h
  3. Vortex Genie 2(Thermo Fisher Scientific,目录号:12-812)
  4. 无菌FACS管
  5. 细胞分选仪(BD Biosciences,型号:FACS aria III)
  6. Reichert Bright-Line Hemocytometer(Sigma-Aldrich,目录号:Z359629)
  7. 解剖剪刀(Roboz Surgical Instrument Co.,目录号:RS-5912)
  8. 钳(Roboz Surgical Instrument Co.,目录号:RS-5137)

程序

保持OP9-DL1细胞

  1. 解冻OP9-DL1
    1. 准备OP9-DL1培养基(配方),并将10ml培养基置于15ml锥形管中
    2. 从液氮中解冻准备的细胞瓶(在FBS + 10%DMSO,1ml总体积)通过将管置于37℃水浴中 直到冰融化(约3分钟)
    3. 使用1ml微量移液管将其加入到10ml OP9-DL1培养基中,并在室温下用235rcf离心5分钟。
    4. 小心地取出培养基,只留下细胞沉淀。
    5. 向管中加入10ml新鲜OP9-DL1培养基,用移液管上下移动以除去沉淀
    6. 将该10ml加入到含有5ml OP9-DL1培养基的T75烧瓶中,总计15Bml。
    7. 在37℃,5%CO 2孵育,每3-4天分裂1:3细胞。

  2. 传递OP9-DL1
    注意:OP9-DL1细胞需要每3 - 4分割1:3,取决于 生长动力学(70%-75%汇合或大约0.6-1×10 10个 在T75烧瓶中)。如果OP9-DL1达到高密度,它们必须是 丢弃,因为它们将变成脂肪细胞。
    1. 准备OP9-DL1培养基(配方),并将10ml培养基置于15ml锥形管中
    2. 从T75烧瓶中取出培养基,加入7ml胰蛋白酶
    3. 在37℃,5%CO 2下孵育约3分钟或直到OP9-DL1开始分离。
    4. 小心取出胰蛋白酶,立即加入10 ml OP9-DL1培养基
    5. 用10ml移液管洗涤烧瓶壁,以移除OP9-DL1细胞
    6. 在25℃下离心260分钟×5分钟。
    7. 除去上清液,加入1ml OP9-DL1培养基沉淀
    8. 使用血细胞计数器计数细胞,并适当分裂(1 T75烧瓶应该有大约300,000个细胞)。

  3. 电镀OP9-DL1用于CLP的分化
    1. 准备OP9-DL1培养基(配方),并将10ml培养基置于15ml锥形管中
    2. 从T75烧瓶中取出培养基,加入7ml胰蛋白酶
    3. 在37℃,5%CO 2下孵育约3分钟或直到OP9-DL1开始分离。
    4. 小心取出胰蛋白酶,立即加入10 ml OP9-DL1培养基
    5. 用10ml移液管洗涤烧瓶壁,以移除OP9-DL1细胞
    6. 在25℃下离心260分钟×5分钟。
    7. 除去上清液,加入1ml OP9-DL1培养基沉淀
    8. 使用血细胞计数板和OP9-DL1板以30000细胞/孔的密度在48孔板中在200-300μl中计数细胞。
    9. 允许细胞至少连接到表面6小时。
    10. 向每个孔中加入1×丝裂霉素-C(200-300μl/孔,50μg/ml终浓度),并在37℃,5%CO 2下孵育25分钟。
    11. 向每孔中轻轻加入1ml每孔温热的OP9-DL1培养基以洗去丝裂霉素-C。
    12. 吸出介质并重复步骤C11,3次。
    13. OP9-DL1饲喂层现在已准备好分离CLP。

第二部分。 CLP与ILC的区别

  1. 常见淋巴祖细胞(CLP)分离和免疫细胞化学
    1. 准备包含3毫升FACS缓冲液(FACS缓冲液保持在4℃)的60×15毫米培养皿
    2. 用含有5ml PBS的25G×5/8针制备5ml注射器
    3. 通过切除近端移除鼠标后肢股骨和胫骨 关节有两端骨开。去除股骨 完全与两端切口
    4. 用镊子握住骨头,通过在注射器中缓慢加入5 ml PBS将骨髓(BM)冲出骨头。
    5. 通过用5ml注射器/25G针2x。
    6. 将细胞(8ml)加入15ml锥形管中
    7. 在25℃,260×g离心细胞5分钟。
    8. 重悬在10毫升无菌分选缓冲液在4°C并计数细胞 通过使用血细胞计数器。细胞的浓度取决于制造商 ?抗体染色说明书。这里,测试定义为1×10 6个细胞/测试,并且以200μl/测试的体积进行。
    9. 通过用冰上的CD16/CD32预孵育细胞(1×10 6个细胞/测试)10分钟来阻断。
    10. 使用"抗体"下列出的指定抗体按照制造商说明对细胞表面分子染色。
    11. 在4℃孵育30分钟。
    12. 通过加入3ml无菌分选缓冲液洗涤细胞,并在235 rcf离心5分钟
    13. 重悬在适量的无菌FACS缓冲液中 然后进行到
    14. 在ARIA III上门控和分选CLP为谱系阴性,CD127 + Flt3 + Sca-1 int (参见图1的门控实例) ARIA II 使用70μm喷嘴。将CLPs直接排入1.5 ml的Eppendorf管中 含有补充有20ng/ml IL-7的300μlILC培养基
    15. 在分选后,在300μl的48孔板中平板7,500个CLPs /孔 cDMEM/IL-7(分类应当净大约15,000CLP /动物) 对丝裂霉素C处理的OP9-DL1(见上文第一部分,C节)
    16. 加入300μl新鲜的cDMEM培养基,补充有20ng/ml IL-7和 20ng/ml SCF每三天。在新鲜的OP9-DL1上进行更新 浓度为每6天7,500个细胞/孔。添加细胞因子 根据什么ILC子类型正在调查(即为ILC-2添加 ?20ng/ml IL-33)。
    17. 可以使用分析细胞的ILC 以前报道的细胞表面标记和转录因子(见 参考)早在CLP平板后6天。如果一个 希望将人群极化为ILC2的命运,IL-33 以20ng/ml的浓度加入到培养基中(即,分离CLP) 直接进入含有细胞因子的培养基中)(图1)

      图1.从野生型小鼠的骨髓分离常见淋巴祖细胞(CLPs)的门控策略。 与轮廓区域相邻的数字表示每个门中的单元格百分比。 CLP具体定义为Lin - CD127 + Flt3 + Sca-1 + 。数据代表来自各自两只小鼠的细胞汇集的10个实验。

食谱

  1. FACS缓冲液(500ml)
    480ml磷酸盐缓冲盐水(PBS)
    15ml FBS(最终3%) 5ml 10%叠氮化钠溶液(在100ml去离子水中的10g NaN 3水溶液)
    储存在4°C
  2. 无菌分选缓冲液(500 ml)
    2.5克BSA(最终0.5%) 12.5 HEPES(最终25mM)
    485 ml PBS
  3. ILC媒体
    DMEM高血糖
    10%FBS
    50μM2-巯基乙醇 1%青霉素 - 链霉素 1mM丙酮酸钠 1个非必需氨基酸
    20mM HEPES(pH7.4) IL-7(20ng/ml) SCF(50ng/ml)
  4. OP9-DL1媒体
    Alpha-MEM
    20%FBS
    1%青霉素 - 链霉素

致谢

这项工作得到美国国家卫生研究院的支持。该方案从参考文献修改(Seehus等人,2015)。

参考文献

  1. Seehus,C.R.,Aliahmad,P.,de la Torre,B.,Iliev,I.D.,Spurka,L.,Funari,V.A.and Kaye,J。(2015)。 先天淋巴细胞的发育需要共同的先天淋巴细胞祖细胞的TOX依赖性产生。 a> Nat Immunol 16(6):599-608
  2. Wong,SH,Walker,JA,Jolin,HE,Drynan,LF,Hams,E.,Camelo,A.,Barlow,JL,Neill,DR,Panova,V.,Koch,U.,Radtke, ,CS,Hwang,YY,Fallon,PG和McKenzie,AN(2012)。 转录因子RORalpha对于黑素细胞发育至关重要。 Nat免疫 > 13(3):229-236。
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引用:Seehus, C. and Kaye, J. (2016). In vitro Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells. Bio-protocol 6(6): e1770. DOI: 10.21769/BioProtoc.1770.
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