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Ex vivo Natural Killer Cell Cytotoxicity Assay
自然杀伤细胞的细胞毒性体外测定   

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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 “gold standard” chromium release assay for measuring the target cell killing capacity of NK cells. Key features of this cytotoxicity assay are that it is performed with sorted NK cells as the effectors and any Major Histocompatibility Class I (MHC-I)-low or deficient tumor cell line can be used as the target cells.

Keywords: Cytotoxicity(细胞毒性), Natural killer cells(自然杀伤细胞), Tumors(肿瘤), Preclinical studies(临床前研究), Innate immunity(先天性免疫)

Materials and Reagents

  1. Lympholyte-M (Cedarlane, catalog number: CL5035 )
  2. RPMI 1640 (Hyclone, catalog number: SH300027.01
  3. Chromium-51 (PerkinElmer, catalog number: NEZ030001MC )
  4. Poly (I:C) (Sigma-Aldrich, catalog number: P1530 )
  5. Source of mouse spenocytes: C57Bl/6 mice (Charles Rivers, strain code: 0 27 )
  6. NK sensitive target cell lines: YAC-1, ATCC, TIB 160
  7. 1x sterile PBS (Hyclone, catalog number: 21-031-CV )
  8. Running buffer (Miltenyi Biotech, catalog number: 130-091-221 )
  9. Washing buffer (Miltenyi Biotech, catalog number: 130-092-987 )
  10. AutoMACS pro columns (Miltenyi Biotech, catalog number: 130-021-101 )
  11. AutoMACS buffer (see Recipes)
  12. Complete RPMI (see Recipes)
  13. NK cell media (see Recipes)

Equipment

  1. 50 ml tubes (BD Biosciences, Falcon®, catalog number: 352098 )
  2. 15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096 )
  3. 96 V well plates (Corning, Costar®, catalog number: 3894 )
  4. AutomacsPro Separator (Miltenyi biotech, model: 130-092-545 )
  5. Gamma counter (PerkinElmer, model: 2470 )
  6. Incubator (5% CO2, 37 °C) (Sanyo)
  7. Centrifuge (when parameters of brakes are unspecified, maxmal acceleration and deceleration are used) (Thermo Fisher Scientific, model: ST40R )
  8. Dissection instruments (small forceps, scissors)
  9. Cell strainers 70 μm (Thermo Fisher Scientific, catalog number: 22363548 )
  10. DX5 (CD49b) microbeads (Miltenyi Biotech, catalog number: 130-052-501 )

Procedure

  1. In vivo stimulation of NK cells
    1. NK cells need to be stimulated in vivo in order to be able to kill. Poly (I: C) (TLR3 agonist) injection is the gold standard method of activating NK cell killing ability.
    2. 3 C57Bl/6 mice (6-8 weeks of age, each weighing approximately 20 g) are usually sufficient in order to get enough NK cells. However, for any in vivo treatment (i.e. virus infection) that might result in lymphopenia or lymphocyte migration to the periophery, 4-5 mice may be needed.
    3. Inject mice intraperitoneal (i.p.) with 150 μg Poly (I: C) (stored at -20 °C, stock is 10 mg/ml) diluted in 1x PBS (15 μl stock + 185 μl 1x PBS) 18 h before euthanizing the mice (e.g. inject at 2:00 PM if you plan on euthanizing mice at 8:00 AM the following day).
    4. Prepare dissection instruments harvesting splencoytes.

  2. Harvest splenocytes
    1. 1 h before starting assay, remove Lympholyte from 4 °C. Lympholyte needs to be used at room temperature and protected from 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 cell strainer over 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 red spleen are observed). Filter again with 10 ml of 1x PBS using the same filter if needed. Spin tubes containing splenocytes 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. Carry-over of small amounts of lympholyte and PBS layers are acceptable because of washing procedure in steps 7 and 8. You can combine mice treatments here (i.e. 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. During spin, harvest target cells. Discard supernatant.

  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 cells or less) and mix well. Incubate for 15 min at 4 °C. During incubation, start target cell labeling with chromium.
    3. Add 10 ml of AutoMACS buffer to stop DX5 microbead 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 row A, negative fraction in row B, positive fraction in row C) of 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.
      Note: “Input” = tube which contain cells to be sorted; “negative fraction” = eluate after sort containing DX5- non-NK cells; “positive fraction” = eluate after sort containing DX5+ NK cells.
    7. Select program: Possel with a quick rinse (qrinse) between each tube and rinse after the last tube. Start the sort. It will take approximately 5-7 min per sort with a 2 min qrinse in between.
    8. After the sort, count the number of cells in the positive fraction tube (2 ml total volume) and determine the cell concentration. Spin down as in step B-7. During spin, prepare 96 V-well plate (add 100 μl NK cell medium to all wells that need it: 3 minimum release wells, and all wells containing 25, 12 and 6 E: T ratios).
    9. Resuspend sorted NK cells at concentration of 1.5 x 106 cells/ml in NK cell medium.
    10. Plate NK effector cells in 100 μl for the top two ratios (50:1 and 25:1), then dilute 2 fold downwards to 12:1 and 6:1 E: T ratios). Scheme: each 50:1 E: T wells contain 1.5 x 105 NK cells; each 25:1 E: T wells contain 7.5 x 104 NK cells; each 12:1 E: T wells contain 3.75 x 104 NK cells; each 6:1 E: T wells contain 1.875 x 104 NK cells .
      Note: All treatments and controls are plated in triplicate wells in 100 μl.
    11. Overlay effector cells with target cells (as prepared in step IV) from a solution of 30,000 cells/ml (3,000 cells/well) in 100 μl.
    12. Add 100 μl of 10x SDS to maximal release wells.
    13. Incubate 4 h in 37 °C, 5% CO2. Spin plate at 500 x g and 4 °C, transfer 100 μl of supernatant to test tubes for CPM counting. Proceed to gamma counter previously calibrated for 51Cr.
    14. % Release measurement.
      (experimental release-average of minimal release)/(average of minimal release-average of maximal release) x 100
    15. Calculate % for each well.

  4. Target cell labeling
    1. Harvest YAC-1 target cells during last spin before the addition of the DX5 microbeads.
      Note: YAC-1 target cells are grown in cRPMI and are non-adherent. They should be passaged for 1.5 weeks prior to use in killing assay.
    2. Start the 51Cr labeling during the 15 min incubation of the splenocytes with the DX5 microbeads.
    3. The 1 h incubation should be done while you are plating your NK effector cells.
    4. Wash three times (twice in cRPMI, final wash in NK cell media), count and resuspend cells at a 30,000 cells/ml concentration.

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

Acknowledgments

This protocol was adapted from the following paper: Patel et al. (2010). 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. Patel, R., Belanger, S., Tai, L. H., Troke, A. D. and Makrigiannis, A. P. (2010). Effect of Ly49 haplotype variance on NK cell function and education. J Immunol 185(8): 4783-4792.

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。 该方案将描述用于测量NK细胞的靶细胞杀伤能力的"金标准"铬释放测定。 该细胞毒性测定的主要特征是其使用分选的NK细胞作为效应物进行,任何主要组织相容性I类(MHC-1) - 低或缺陷的肿瘤细胞系可以用作靶细胞。

关键字:细胞毒性, 自然杀伤细胞, 肿瘤, 临床前研究, 先天性免疫

材料和试剂

  1. 淋巴细胞-M(Cedarlane,目录号:CL5035)
  2. RPMI 1640(Hyclone,目录号:SH300027.01)
  3. Chromium-51(PerkinElmer,目录号:NEZ030001MC)
  4. 聚(I:C)(Sigma-Aldrich,目录号:P1530)
  5. 小鼠脾细胞来源:C57Bl/6小鼠(Charles Rivers,菌株代码:027)
  6. NK敏感性靶细胞系:YAC-1,ATCC,TIB 160
  7. 1×无菌PBS(Hyclone,目录号:21-031-CV)
  8. 运行缓冲液(Miltenyi Biotech,目录号:130-091-221)
  9. 洗涤缓冲液(Miltenyi Biotech,目录号:130-092-987)
  10. AutoMACS pro柱(Miltenyi Biotech,目录号:130-021-101)
  11. AutoMACS缓冲区(请参阅配方)
  12. 完成RPMI(参见配方)
  13. NK细胞培养基(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. 96 V孔板(Corning,Costar ,目录号:3894)
  4. AutomacsPro Separator(Miltenyi biotech,型号:130-092-545)
  5. γ计数器(PerkinElmer,型号:2470)
  6. 培养箱(5%CO 2,37℃)(Sanyo)
  7. 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)
  8. 解剖器械(小钳子,剪刀)
  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. 体内刺激NK细胞
    1. NK细胞需要在体内被刺激以便能够杀死。 Poly(I:C)(TLR3激动剂)注射是激活NK细胞杀伤能力的金标准方法
    2. 3 C57Bl/6小鼠(6-8周龄,每只重约20g)通常足以获得足够的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(即病毒感染),可能需要4-5只小鼠。
    3. 在安乐死小鼠之前18小时腹膜内(ip)注射用1x PBS(15μl储备液+185μl1×PBS)稀释的150μgPoly(I:C)(储存在-20℃,储备液为10mg/ml) (例如注入 如果您计划在第二天上午8:00安乐死小鼠,则在下午2:00。)
    4. 准备解剖器械收获脾脏
  2. 收获脾细胞
    1. 在开始测定前1小时,从4℃除去淋巴细胞。 淋巴细胞需要在室温下使用,避免光照(在生物安全柜下关闭灯泡处理)。
    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70微米的细胞过滤器。 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脾细胞过滤器超过50ml管,用10ml的1x PBS冲洗两次(我经常冲洗过滤器的下面,如果可见红色脾脏块)。如果需要,使用相同的过滤器再次用10ml 1×PBS进行过滤。旋转管含有脾细胞,500×g/g,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。因为步骤7和8中的洗涤程序,可以接受少量的淋巴细胞和PBS层。您可以在这里组合小鼠治疗(所有相同的治疗方法)。
    7. 用1x PBS填充50ml管(首先洗涤以除去过量的淋巴细胞)。向下旋转500 x g ,5分钟4°C
    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。 在旋转期间,收获目标细胞。 弃去上清液。

  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1×10 8个细胞或更少),并充分混合。 在4℃孵育15分钟。 孵育期间,用铬开始靶细胞标记。
    3. 加入10毫升AutoMACS缓冲液停止DX5微珠孵化。 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. 放置管架(A行中的输入,B行中的负部分,C行中的正部分)。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 ml管,5支架的15 ml管和5支6支架的流式细胞仪管。
      注意:"Input"=包含要排序的单元格的管; "负分数"=含有DX5-非NK细胞的分选后的洗脱液; "阳性分数"=含有DX5 + NK细胞的分选后的洗脱液。
    7. 选择程序: Possel ,在每个试管之间快速冲洗(qrinse),并在最后一个试管之后冲洗。开始排序。每次排序需要大约5-7分钟,中间有2分钟的时间
    8. 分选后,计数阳性部分管中的细胞数(2ml总体积)并测定细胞浓度。如步骤B-7中那样旋转。在旋转过程中,准备96 V孔板(添加100微升NK细胞培养基到所有需要它的孔:3个最小释放孔,所有孔含有25,12和6 E:T比率)。
    9. 在NK细胞培养基中以1.5×10 6个细胞/ml的浓度重悬浮分选的NK细胞。
    10. 平板NK效应细胞在100微升的顶部两个比例(50:1和25:1),然后稀释2倍下降到12:1和6:1的E:T比率)。方案:每个50:1 E:T孔包含1.5×10 5个NK细胞;每个25:1E:T孔含有7.5×10 4个NK细胞;每个12:1E:T孔含有3.75×10 4个NK细胞;每个6:1E:T孔含有1.875×10 4个NK细胞。
      注意:所有处理和对照在一式三份的孔中接种在100μl中。
    11. 用来自在100μl中的30,000个细胞/ml(3,000个细胞/孔)的溶液覆盖具有靶细胞(如步骤IV中制备的)的效应细胞。
    12. 向最大释放孔中加入100μl10×SDS
    13. 在37℃,5%CO 2中孵育4小时。在500×g和4℃下旋转板,将100μl上清液转移到试管中用于CPM计数。继续先前为 51 Cr。校准的伽马计数器。
    14. %释放测量。
      (最小释放的实验释放平均值)/(最小释放的平均值 - 最大释放的平均值)×100
    15. 计算每口井的%。

  4. 靶细胞标记
    1. 在添加DX5微珠之前的最后一次旋转期间收获YAC-1靶细胞。
      注意:YAC-1靶细胞在cRPMI中生长并且是非粘附的。 在用于杀灭测定之前,应将其传代1.5周。
    2. 在用DX5微珠孵育脾细胞15分钟时开始 51 Cr标记。
    3. 孵育1小时应该在你电泳你的NK效应细胞
    4. 洗涤三次(在cRPMI中两次,在NK细胞培养基中最后洗涤),计数并以30,000细胞/ml浓度重悬细胞。

食谱

  1. AutoMACS缓冲液在500ml
    中 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

致谢

该协议改编自以下论文:Patel等人(2010)。 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. Patel,R.,Belanger,S.,Tai,L.H.,Troke,A.D.and Makrigiannis,A.P。(2010)。 Ly49单倍型差异对NK细胞功能和教育的影响 J Immunol 185(8):4783-4792。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tai, L., Souza, C. T., Makrigiannis, A. P. and Auer, R. A. (2013). Ex vivo Natural Killer Cell Cytotoxicity Assay. Bio-protocol 3(16): e863. DOI: 10.21769/BioProtoc.863.
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