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Natural Killer Cell Transfer Assay
自然杀伤细胞迁移试验

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Abstract

Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the adoptive transfer of purified NK cells into NK cell-deficient tumor bearing mice in order to establish the intrinsic functionality of NK cells.

Keywords: Innate immunology(先天性免疫), Natural killer cells(自然杀伤细胞), Adoptive transfer assays(过继转移实验), Cell intrinsic(细胞内源)

Materials and Reagents

  1. Lympholyte-M (Cedarlane, catalog number: CL5035 )
  2. Source of mouse NK cells: C57Bl/6 mice (Charles Rivers, strain code: 027 )
  3. NK-deficient mice (Jackson Labs, strain name: B6.129S4-Il2rgtm1Wjl/J, strain number: 003174 )
  4. Gluteraldehyde
  5. Potassium ferrocyanide
  6. Potassium ferricyanide
  7. Bovine serum albumin (BSA)
  8. 100x non-essential amino acids
  9. Fetal bovine serum (FBS)
  10. Sodium pyruvate
  11. Deoxycholate
  12. B16F10lacZ melanoma tumor cells, grown in complete DMEM (Dr. K. Graham, London Regional Health Centre, London ON)
  13. 1x sterile PBS (Hyclone, catalog number: 21-031-CV )
  14. X gal (Bioshop Canada Inc., catalog number: XGA001-1 )
  15. Running buffer (Miltenyi Biotech, catalog number: 130-091-221 )
  16. Washing buffer (Miltenyi Biotech, catalog number: 130-092-987 )
  17. AutoMACS pro columns (Miltenyi Biotech, catalog number: 130-021-101 )
  18. RPMI 1640 (Hyclone, catalog number: SH300027.01 )
  19. AutoMACS buffer (see Recipes)
  20. Complete RPMI (see Recipes)
  21. NK cell media (see Recipes)
  22. LacZ staining solutions (see Recipes)

Equipment

  1. 50 ml tubes (BD Biosciences, Falcon®, catalog number: 352098 )
  2. 15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096 )
  3. Insulin syringe 27G1/2” (Terumo Medical Corporation, catalog number: SS05M2713 )
  4. AutomacsPro Separator (Miltenyi biotech, model: 130-092-545 )
  5. Incubator (5% CO2, 37 °C) (Sanyo)
  6. Centrifuge (when parameters of brakes are unspecified, maximal acceleration and deceleration are used), (Thermo Fisher Scientific, model: ST40R )
  7. Dissection instruments (small forceps, scissors)
  8. Light microscope (Leica)
  9. Cell strainers 70 µm (Thermo Fisher Scientific, catalog number: 22363548 )
  10. DX5 (CD49b) microbeads (Miltenyi Biotech, catalog number: 130-052-501 )

Procedure

  1. Treatment of donor mice – mouse model of surgical stress
    1. Donor mice (6-8 weeks of age, each weighing approximately 20 g) were treated with abdominal left nephrectomy (surgical stress) 18 h prior to harvesting spleen NK cells for transfer into recipient mice.
    2. 3 C57Bl/6 mice are usually sufficient in order to get 2.0 x 106 NK cells. However, for any in vivo treatment (i.e. in vivo virus infection) that might result in lymphopenia or lymphocyte migration to the periophery, more mice may be needed.
    3. Prepare dissection instruments for harvesting splenocytes.

  2. Harvest donor splenocytes
    1. 1 h before starting the harvest, remove Lympholyte from 4 °C. Lympholyte needs to be used at room temperature and protected for light (handle under biosafety cabinet with lights off).
    2. Prepare one 50 ml tube and two 15 ml tubes for each spleen. Place a 70 μM cell strainer on each opened 50 ml tube. Prime each strainer with 1 ml of cold 1x PBS. Add 5 ml of Lympholyte into each 15 ml tube (protect tubes from light).
      Note: 1 spleen will need two 15 ml tubes, each containing 5 ml Lympholyte.
    3. Euthanize mice by cervical dislocation, remove spleen and place on 70 μM strainer
    4. Crush 1 spleen on a 70 μM cell strainer over a 50 ml tube, rinse twice with 10 ml of 1x PBS (I often rinse the underside of the filters as well if visible clumps of spleen are observed). Filter again with 10 ml of 1x PBS using the same filter if needed. Spin at 500 x g, 5 min, 4 °C.
    5. Discard supernatant and resuspend splenocyte pellet in 10 ml 1x PBS. Carefully layer 5 ml of resuspended splenocytes on top of the Lympholyte layer (5 ml of the 1st 15 ml tubes, then the remaining 5 ml on the 2nd 15 ml tube). Spin 1,500 x g, 15 min, room temperature, acceleration at 1, deceleration at 2 (minimal speed).
    6. Carefully pipet lymphocyte layer (blurry interface layer between Lympholyte at the bottom and PBS on top) and transfer to a new 50 ml tube. You can combine mice treatments here (e.g. all the same treatments together).
    7. Fill 50 ml tube with 1x PBS (first wash to remove excess Lympholyte). Spin down 500 x g, 5 min, 4 °C.
    8. Discard supernatant. Resuspend pellet in 10 ml AutoMACS buffer. Spin down as in step B-7. Discard supernant.

  3. NK cell sort (with DX5 microbeads)
    1. Resuspend splenocytes pellet in 300 μl AutoMACS buffer per spleen (we usually pool 3 spleens per tube).
    2. Add 100 μl of DX5 microbeads per spleen (manufacturer recommends 100 μl beads volume for 1 x 108 or less cells) and mix well. Incubate for 15 min at 4 °C.
    3. Add 10 ml of AutoMACS buffer to stop DX5 incubation. Spin as in step B-7.
    4. Discard supernatant. Resuspend pellet in 500 μl AutoMACS buffer per spleen (e.g. 1.5 ml for 3 pool spleens).
    5. Proceed to sort. Turn AutoMACS Pro sorter on during last spin and do a rinse before starting.
    6. Place tubes (input in A, negative fraction in B, positive fraction in C) in rack holder. The size of the tubes used for sort depends on the AutoMACS rack holder used. Usually a standard 50 ml tube for the 3 holder rack, 15 ml tube for 5 holder rack, and 5 ml flow cytometry tubes for 6 holder rack. “Negative fraction” denotes the eluate after the sort, which contains DX5- non-NK cells. “Positive fraction” denotes the eluate after the sort, which contains DX5+ NK cells.
    7. Select program: Possel with a quick rinse (qrinse) between each tube and rinse after the last tube. Start the sort.
    8. After the sort, count the number of DX5+ cells in the positive fraction tube (2 ml total volume) and determine the cell concentration. Spin down. NK cell purity as determined by flow cytometry (DX5+, TCRb-) is usually > 90%.

  4. NK cell transfer and tumor injection
    1. Resuspend 1.0 x 106 DX5+ NK cells in sterile 1x PBS cells and inject via tail vein injection (100 μl total volume) into NK-deficient mice.
    2. 1 h post NK cell injection, 3 x 105 B16lacZ tumor cells resuspended in 100 μl serum free DMEM (greater than 90% viability as determined by trypan blue), inject via tail vein injection (100 μl total volume) into the same NK-deficient mice.
      Note: For mouse model of surgical stress experiments, recipient NK-deficient mice received NK cells from surgically stressed and untreated control donor mice.
    3. Allow mice to survive for 3 days post tumor cell injection.

  5. B16lacZ lung tumor quantification
    1. 3 days post NK and tumor cell injection, euthanize recipient mice and extract all 5 lobes of the lungs.
    2. To extract lungs, expose the thorax by cutting through the skin and subcutaneous layer along the ventral midline of the chest cavity of the mouse. Next, make lateral incisions through skin and tissue on each side up to the neck of the mouse. Then, gently grasp lungs with surgical forceps and gently dissected by snipping away the connective tissue above and below the lungs.
    3. Rinse lungs in Phosphate buffer (pH 7.3).
    4. Place lungs into scintillating vial containing 5 ml ice-cold Phosphate buffer (pH 7.3). Keep on ice until next step.
    5. Pour out Phosphate buffer, being careful not to lose lungs. Fix lungs for 20 min by adding in fixative solution (8 ml/vial).
    6. Wash twice for 10 min in wash buffer solution (8 ml/vial).
    7. Stain with X gal overnight at 37 °C (12-18 h–2 ml/vial).
    8. Wash once with wash buffer (5 ml/vial for 10 min), then add fresh wash buffer (10 ml/vial) and store at 4 °C overnight. Staining will intensify.
    9. Aspirate wash buffer and add 15 ml/vial of 10% buffered Formalin for preservation.
    10. Quantify lung metastases with light microscope.
    11. See Figure 1: Representative lung picture depicting B16lacZ lung metastases at day 3 post tumor cell intravenous injection is shown.


      Figure 1. Representative lung pictures showing B16lacZ lung tumor metastases at day 3 post-tumor cell intravenous injection

Recipes

  1. Automacs buffer in 500 ml
    PBS
    2.5 g Bovine Serum Albumin
    2 ml of 5 mM EDTA
  2. Complete RPMI in 500 ml
    500 ml of RPMI-1640
    50 ml Heat-inactivated Fetal Bovine Serum
    5 ml of Pencillin-Streptomycin 10,000 U each/ml
  3. NK cell media
    500 ml of cRPMI
    5 ml 1 M HEPES
    5 ml 100 mM Sodium Pyruvate
    5 ml 100x Non-Essential Amino Acids
    0.5 ml of 2-mercaptoethanol for final concentration of 5 x 10-5 M
  4. LacZ staining working solutions
    1. 0.1 M Phosphate buffer in 5 L at pH 7.3
      15.87 g Sodium Phosphate Monobasic (MW 137.99)
      54.67 g Sodium Phosphate Dibasic (MW 141.96)
      Dissolve into 5 L of dH2O
    2. Fixative solution in 900 ml
      45 ml 100 mM EGTA (pH 7.3)
      1.8 ml 1 M Magnesium Chloride
      846 ml 0.1 M Phosphate buffer (pH 7.3)
      Prepare the stock solution without gluteraldehyde
      Aliquot 1.8 ml of 25% gluteraldehyde into 223.3 ml of stock when you are ready to use
      This will allow you to prepare 225 ml of fresh fixative solution.
    3. Wash buffer in 3.6 L
      7.2 ml 1 M Magnesium Chloride
      36 ml 1% Deoxycholate
      36 ml 2% Nonidet-P40
      3,520.8 ml 0.1 M Phosphate buffer (pH 7.3)
    4. 25 mg/ml X gal stock
      1 g of X gal in 40 ml of DMSO
    5. X gal stain in 1 L
      40 ml of 25 mg/ml X gal stock
      2.12 g Potassium Ferrocyanide (MW 422.2)
      1.64 g Potassium Ferricyanide (MW 329.2)
      960 ml Wash buffer

Acknowledgments

This protocol was adapted from the following paper: Kirstein et al. (2009). This study was supported by Canadian Cancer Society Research Institute, Ontario Regional Biotherapeutics (ORBiT) program, Private Donor (D.H.) Ottawa Hospital Foundation, (R.A. Auer) and Fonds de Recherche Sante Quebec (L.-H. Tai and S. Belanger).

References

  1. Kirstein, J. M., Graham, K. C., Mackenzie, L. T., Johnston, D. E., Martin, L. J., Tuck, A. B., MacDonald, I. C. and Chambers, A. F. (2009). Effect of anti-fibrinolytic therapy on experimental melanoma metastasis. Clin Exp Metastasis 26(2): 121-131.

简介

天然杀伤(NK)细胞是细胞毒性淋巴细胞,其构成先天性免疫系统的主要组分。 通过NK细胞对恶性和病毒感染的细胞的宿主的免疫监视导致直接的细胞毒性和细胞因子的产生,以增强免疫应答。 该方案将描述纯化的NK细胞过继转移到NK细胞缺陷的荷瘤小鼠中以建立NK细胞的内在功能。

关键字:先天性免疫, 自然杀伤细胞, 过继转移实验, 细胞内源

材料和试剂

  1. 淋巴细胞-M(Cedarlane,目录号:CL5035)
  2. 小鼠NK细胞来源:C57Bl/6小鼠(Charles Rivers,菌株代码:027)
  3. NK缺陷小鼠(Jackson Labs,菌株名称:B6.129S4-II2rg tm1Wi1/J,菌株号:003174)
  4. 戊二醛
  5. 亚铁氰化钾
  6. 铁氰化钾
  7. 牛血清白蛋白(BSA)
  8. 100x非必需氨基酸
  9. 胎牛血清(FBS)
  10. 丙酮酸钠
  11. 脱氧胆酸盐
  12. B16F10lacZ黑素瘤肿瘤细胞,在完全DMEM(Dr.G.Graham,London Regional Health Center,London ON)中生长。
  13. 1×无菌PBS(Hyclone,目录号:21-031-CV)
  14. X gal(Bioshop Canada Inc.,目录号:XGA001-1)
  15. 运行缓冲液(Miltenyi Biotech,目录号:130-091-221)
  16. 洗涤缓冲液(Miltenyi Biotech,目录号:130-092-987)
  17. AutoMACS pro柱(Miltenyi Biotech,目录号:130-021-101)
  18. RPMI 1640(Hyclone,目录号:SH300027.01)
  19. AutoMACS缓冲区(请参阅配方)
  20. 完成RPMI(参见配方)
  21. NK细胞培养基(参见配方)
  22. LacZ染色溶液(参见配方)

设备

  1. 50ml管(BD Biosciences,Falcon ,目录号:352098)
  2. 15ml管(BD Biosciences,Falcon ,目录号:352096)
  3. 胰岛素注射器27G1/2"(Terumo Medical Corporation,目录号:SS05M2713)
  4. AutomacsPro Separator(Miltenyi biotech,型号:130-092-545)
  5. 培养箱(5%CO 2,37℃)(Sanyo)
  6. 离心机(未指定制动器参数时,使用最大加速度和减速度),(Thermo Fisher Scientific,型号:ST40R)
  7. 解剖器械(小钳子,剪刀)
  8. 光学显微镜(Leica)
  9. 细胞过滤器70μm(Thermo Fisher Scientific,目录号:22363548)
  10. DX5(CD49b)微珠(Miltenyi Biotech,目录号:130-052-501)

程序

  1. 供体小鼠的治疗 - 小鼠手术应力模型
    1. 在收获脾NK细胞以转移到受体小鼠中之前18小时,用腹部左肾切除术(手术应激)处理供体小鼠(6-8周龄,各自重约20g)。
    2. 3 C57Bl/6小鼠通常足以获得2.0×10 6个NK细胞。 然而,对于可能导致淋巴细胞减少或淋巴细胞迁移到周围的任何体内治疗(即 体内病毒感染) 需要。
    3. 准备解剖器械收获脾细胞
  2. 收获供体脾细胞
    1. 在开始收获前1小时,从4℃除去淋巴细胞。 淋巴细胞需要在室温下使用并保护光线(在生物安全柜下关灯)。
    2. 为每个脾准备一个50ml管和两个15ml管。在每个打开的50ml管上放置70μM细胞过滤器。用1ml冷的1×PBS对每个过滤器进行预处理。加入5ml的淋巴细胞到每个15毫升管(保护管从光)。
      注意:1只脾脏需要两根15ml的管,每根管含有5ml的淋巴细胞。
    3. 安乐死小鼠颈椎脱位,取出脾脏,并置于70μM滤网上
    4. 粉碎1脾在70μM细胞过滤器在50ml管,用10毫升1×PBS冲洗两次(我经常冲洗过滤器的下面,如果可见的脾脏的观察)。如果需要,使用相同的过滤器再次用10ml 1×PBS进行过滤。在500摄氏度下旋转,5分钟,4℃
    5. 弃去上清液并将脾细胞沉淀重悬于10ml 1x PBS中。小心地将5mL重悬浮的脾细胞层置于淋巴层上(5ml第一个15ml管,然后剩余5ml上的第二个15ml管) )。旋转1,500分钟,室温,15分钟,加速度为1,减速度为2(最小速度)。
    6. 小心吸取淋巴细胞层(底部淋巴细胞和顶部PBS之间的模糊界面层),并转移到新的50ml管中。您可以在这里结合小鼠治疗(例如所有相同的治疗)。
    7. 用1x PBS填充50ml管(首先洗涤以除去过量的淋巴细胞)。 在500℃下旋转5分钟,4℃。
    8. 弃去上清液。 重悬在10ml AutoMACS缓冲液中的沉淀。 如步骤B-7中那样旋转。 舍弃上层。

  3. NK细胞分选(用DX5微珠)
    1. 重悬脾细胞沉淀在每个脾脏300微升AutoMACS缓冲液(我们通常每管3个脾)。
    2. 每个脾脏添加100微升DX5微珠(制造商建议100微升珠体积为1 x 10 8或更少的细胞)并混匀。 在4℃孵育15分钟。
    3. 加入10毫升AutoMACS缓冲液停止DX5孵化。旋转,如步骤B-7
    4. 弃去上清液。在500μlAutoMACS缓冲液/脾(例如对于3个池脾脏1.5ml)中重悬沉淀物。
    5. 继续排序。在最后一次旋转期间打开AutoMACS Pro排序器,并在开始之前冲洗。
    6. 将管(输入A,阴性部分在B,阳性部分在C)放在架子里。用于分拣的管的尺寸取决于所使用的AutoMACS机架。通常为3支架的标准50毫升管,5支架的15毫升管和5毫升6支架的流式细胞仪管。 "阴性部分"表示分选后的洗脱液,其含有DX5 +非NK细胞。 "阳性分数"表示分选后的洗脱液,其含有DX5 + NK细胞。
    7. 选择程序:在每个管之间快速冲洗(qrinse),并在最后一个管之后冲洗。开始排序。
    8. 分选后,计数阳性分份管中的DX5 +细胞数(2ml总体积),并测定细胞浓度。向下旋转。通过流式细胞术测定的NK细胞纯度 (DX5 + ,TCRb - )通常> 90%。

  4. NK细胞转移和肿瘤注射
    1. 在无菌1×PBS细胞中重悬1.0×10 6个DX5 + sup/+ NK细胞,并通过尾静脉注射(100μl总体积)注射到NK缺陷小鼠中。
    2. 在NK细胞注射后1小时,将3×10 5个B16lacZ肿瘤细胞重悬于100μl无血清DMEM(通过台盼蓝测定大于90%生存力)中,通过尾静脉注射(100μl总计 体积)进入相同的NK缺陷小鼠 注意:对于手术应激实验的小鼠模型,受体NK缺陷小鼠从手术应激和未治疗的对照供体小鼠接受NK细胞。
    3. 允许小鼠在肿瘤细胞注射后存活3天
  5. B16lacZ肺肿瘤定量
    1. NK和肿瘤细胞注射后3天,安乐死受体小鼠,并提取所有5个肺叶
    2. 为了提取肺,通过穿过皮肤和皮下层沿着小鼠胸腔的腹侧中线切开暴露胸部。 接下来,通过每侧的皮肤和组织进行横向切口直到小鼠的颈部。 然后,用手术钳轻轻抓住肺,轻轻地摘除肺上方和下方的结缔组织。
    3. 在磷酸盐缓冲液(pH 7.3)中冲洗肺。
    4. 将肺放入含有5ml冰冷的磷酸盐缓冲液(pH 7.3)的闪烁瓶中。 保持冰上直到下一步。
    5. 倒出磷酸盐缓冲液,小心不要失去肺。 通过加入固定溶液(8ml /瓶)将肺固定20分钟
    6. 在洗涤缓冲液(8ml /瓶)中洗涤两次,每次10分钟
    7. 在37℃下用Xgal染色过夜(12-18小时-2ml /小瓶)
    8. 用洗涤缓冲液洗涤一次(5ml /瓶10分钟),然后加入新鲜的洗涤缓冲液(10ml /瓶)并在4℃下储存过夜。 染色将加剧。
    9. 吸出洗涤缓冲液,加入15 ml /瓶10%缓冲福尔马林保存。
    10. 用光学显微镜定量肺转移。
    11. 参见图1:显示了在肿瘤细胞静脉内注射后第3天描绘B16lacZ肺转移的代表性肺图。


      图1.显示在肿瘤细胞静脉内注射后第3天的B16lacZ肺肿瘤转移的代表性肺图片

食谱

  1. Automacs缓冲液在500 ml
    PBS
    2.5g牛血清白蛋白
    2ml 5mM EDTA
  2. 在500 ml完成RPMI
    500ml RPMI-1640
    50 ml热灭活胎牛血清
    5ml青霉素 - 链霉素10,000U/ml/ml
  3. NK细胞培养基
    500 ml cRPMI
    5 ml 1 M HEPES
    5ml 100mM丙酮酸钠 5 ml 100x非必需氨基酸
    0.5ml 2-巯基乙醇,最终浓度为5×10 -5 M
  4. LacZ染色工作溶液
    1. 0.1M磷酸盐缓冲液,5L pH7.3 15.87g磷酸二氢钠(MW 137.99) 54.67g磷酸二氢钠(MW 141.96)
      溶解到5L dH 2 O中
    2. 900 ml固定溶液
      45ml 100mM EGTA(pH7.3)
      1.8ml 1M氯化镁 846ml 0.1M磷酸盐缓冲液(pH7.3) 准备没有戊二醛的储备溶液
      当您准备使用时,将1.8毫升25%的戊二醛分装到223.3毫升的原液中。
      这将允许您准备225毫升新鲜固定溶液。
    3. 洗涤缓冲液3.6 L
      7.2ml 1M氯化镁
      36ml 1%脱氧胆酸盐 36ml 2%Nonidet-P40
      3,520.8ml 0.1M磷酸盐缓冲液(pH7.3)
    4. 25mg/ml X gal母体
      1克X gal在40毫升DMSO中的溶液
    5. X gal染色在1 L
      40ml 25mg/ml X gal母体
      2.12g氰亚铁酸钾(MW 422.2)
      1.64g氰铁酸钾(MW 329.2)
      960 ml洗涤缓冲液

致谢

该协议改编自以下论文:Kirstein等人(2009)。 这项研究由加拿大癌症协会研究所,安大略省区域生物治疗(ORBiT)计划,私人捐助者(D.H.) 渥太华医院基金会(R.A. Auer)和魁北克省魁北克省医院(L.-H. Tai和S. Belanger)。

参考文献

  1. Kirstein,J.M.,Graham,K.C.,Mackenzie,L.T.,Johnston,D.E.,Martin,L.J.,Tuck,A.B.,MacDonald,I.C。和Chambers,A.F。 抗纤维蛋白溶解疗法对实验性黑色素瘤转移的影响临床转移性转移 26(2):121-131。
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引用:Tai, L., Souza, C. T., Makrigiannis, A. P. and Auer, R. A. (2013). Natural Killer Cell Transfer Assay. Bio-protocol 3(16): e864. DOI: 10.21769/BioProtoc.864.
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