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Lung Clearance Assay
肺部清除试验   

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Abstract

Lung clearance assay tests the ability of innate immune cells (mainly NK cells) to eradicate in vivo cells injected via the tail vein of the mice. This assay helps to elucidate the role played by NK cells and their receptors (if the mice are genetically modified) against various human and mouse targets in an in vivo setting (Stern-Ginossar et al., 2008; Halfteck et al., 2009; Tsukerman et al., 2012).

Keywords: NK(NK), In-Vivo-killing(在Vivo杀害), Lung-clearance(肺清除)

Materials and Reagents

  1. Cell labeling dyes
    1. Vybrant CM-DiI (Life Technologies, InvitrogenTM, catalog number: V-22888 )
    2. VybrantDiD (Life Technologies, InvitrogenTM, catalog number: V-22887 )
    3. CellTrace CFSE (Life Technologies, InvitrogenTM, catalog number: C-34554 ) (either CM-DiI or CFSE can be used based on the preferred wave length either FL-1 for CFSE or FL-2 for the CM-DiI)
  2. Other materials
    1. Phosphate buffered saline (PBS)
    2. 1x Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen,Life Technologies, InvitrogenTM, catalog number: 10313-039 )
    3. Fetal bovine serum (Life Technologies, InvitrogenTM, catalog number: 11091-148 )
    4. 40 micron cell strainer (BD Biosciences, Falcon®, catalog number: 352340 )
    5. Mice of interest
    6. NaN3 (Sodium Azide)
    7. Erythrocyte lysis buffer (ELB) (see Recipes)
    8. FACS buffer (see Recipes)
  3. Antibodies
    To demonstrate that the observed effect is specific we recommend to either blocking the relevant receptors, or by depleting NK cells, by using anti-NK1.1 (in case the receptor is present), or by using anti-asialo GM1.
    1. Anti NK1.1 (eBioscience, catalog number: 14-5941-82 )
    2. Anti-Asialo-GM1 (eBioscience, catalog number: 16-6507-39 )
    3. Anti-Mouse CD314 (NKG2D) (eBioscience, catalog number: 14-5873-82 )

Equipment

  1. BD LSR II flow cytometer

Procedure

  1. The tail vein injection requires prior practice, mature male mice are easier to inject. We suggest using 8-12 weeks male mice.
  2. The depletion of NK cells for a period of one day is obtained by I.P (Intra peritoneal) injection of 150 microgram (in up to 250 μl PBS) of anti NK1.1 up to 24 h prior to the experiment.
  3. Note: Not all cell types are suitable for these experiments, small cells are hard to recover, large cells may clog the lungs, some cells are not labeled well etc. Therefore, preliminary experiments using limited amount of animals are strongly recommended. The lines that we tested are: HeLa, PC-3, DU 145, PD1.6 and YAC-1. Since the tail vein injections may vary between different mice even in the same group there is a need for an internal injection control which is usually cells that are resistant to mouse NK cell killing. Label two cell types (experiment and control) according to the manufacturer instructions for the dyes. If the cells used were not in the manufacturer protocol calibration in vitro staining experiments are required. Labeling of HeLa (the control population for the majority of our experiments), Jurkat, 3T3, P3X, PC-3 DU 145 is performed as follows:
    1. Suspend cells at a density of 1 x 106/ml in serum-free culture medium (DMEM or RPMI).
    2. Add 1- 5 μl (from the stock) of the cell-labeling solution per ml of cell suspension. Mix well by gentle pipetting. Do not exceed 5 ml volume in each labeling tube. The differences between CFSE and Vybrant-CM-DiI are, the emission (FL-1/FL-2) and the range of labeled cells (we recommend working with the Vybrant-CM-DiI when possible).
    3. Incubate at 37 °C HeLa (8 min), Jurkat (2 min), 3T3 (15 min), P3X (15 min), PC-3 (30 min) DU 145 (30 min). We recommend incubating in 15 ml tubes covered with aluminum foil.
    4. Centrifuge at 450-515 x g for 5 min.
    5. Remove the supernatant and gently resuspend the cells in warm (37 °C) medium.
    6. Repeat the wash procedure (3-d and 3-e).
  4. Resuspend the cells to a final concentration of 107 cells per ml these cells are to be injected to the mice, 2 x 106cells of each cell type (4 x 106 total) are injected via the tail vein per mouse; the final injection volume is 400 μl.
  5. Pass the cells through a 40 μm cell strainer.
  6. Set aside 400 μl of the cell mixture that will serve as the pre-injection tube. Keep these cells at 37 °C for the duration of the experiment.
  7. Inject the cells into the tail vein of the mice.
  8. Wait for 5 h.
  9. Sacrifice the mice according to ethical regulations, we recommend using CO2.
  10. Harvest the lungs, place them in a 6 well plates with 2 ml ice cold PBS until harvesting the lungs of the entire experiment is completed.
  11. Place a 40 μm cell strainer on top of a 50 ml tube, transfer the lungs into the strainer, transfer the PBS from the 6 well into the tube.
  12. Smash the lungs by using the cylinder/pestle of a 5 ml syringe; add more PBS as required.
  13. After obtaining a homogenous solution centrifuge the tubes at 450-515 x g for 5 min.
  14. Discard supernatant and resuspend cells with 10 ml of ELB (erythrocyte lysis buffer) and keep on ice for 5 min.
  15. Centrifuge the cells at 450-515 x g for 5 min.
  16. Discard supernatant and observe for erythrocyte content (if there is visible amount of red cells in the pellet) repeat steps 15-16, until the red blood cells disappear.
  17. Resuspend the cells in 10 ml of DMEM/RPMI supplemented with 10% FBS (this step eliminates the ELB activity).
  18. Centrifuge the cells at 450-515 x g for 5 min.
  19. Discard supernatant and resuspend cells in 1-2 ml of FACS buffer according topellet size.
  20. Examine the cells using FACS. Begin with the pre-injection sample, set the gates based on forward and side scatter (see figure below), and calibrate the laser intensity. Collect at least 2.5 x 105 cells. Draw a dot plot of FL-4 vs FL-2 (if using DiI) or FL-4 vs FL-1 (if using CFSE).
  21. Read the lung samples. We recommend at collecting least 2.5 x 106 cells per sample
  22. Note: cells can be resuspended in 2% formaldehyde and stored overnight at 4 °C in dark, prior to analysis by flow cytometry.
  23. Flow cytometric analysis (Figure 1): Y axis FL-1 intensity X axis FL-4 intensity. The square frame marks the “experiment” cell population; the dashed square frame marks the “internal control” cell population. The lower left square are lung cells of the mice ( FL-1, FL-4 negative).


    Figure 1. Flow cytometric analysis

  24. To obtain the killing percentage, the amount of experimental cells (NK sensitive) divided by the amount of the control cells (NK insensitive) in the pre-injection group is calculated and this ratio is set up to be the reference control. Accordingly, similar calculation is performed regarding the cells recovered in the lungs 5 h post infection and tumor cell survival is calculated.

Recipes

  1. ELB (erythrocyte lysis buffer)
    NH4Cl 16.4 g
    KHCO3 2 g
    EDTA 0.5 M 400 μl
    2 L ddH2O
    Titrate with HCl to pH 7.2-7.4
  2. FACS buffer
    1x PBS
    0.5% BSA
    0.05% NaN3 (Sodium Azide)

Acknowledgments

The authors would like to acknowledge Noam Stern-Ginossar, Chamutal Gur, Ariella Glasner, and Mazal El-Nekaveh.

References

  1. Halfteck, G. G., Elboim, M., Gur, C., Achdout, H., Ghadially, H. and Mandelboim, O. (2009). Enhanced in vivo growth of lymphoma tumors in the absence of the NK-activating receptor NKp46/NCR1. J Immunol 182(4): 2221-2230.
  2. Stern-Ginossar, N., Gur, C., Biton, M., Horwitz, E., Elboim, M., Stanietsky, N., Mandelboim, M. and Mandelboim, O. (2008). Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D. Nat Immunol 9(9): 1065-1073.
  3. Tsukerman, P., Stern-Ginossar, N., Gur, C., Glasner, A., Nachmani, D., Bauman, Y., Yamin, R., Vitenshtein, A., Stanietsky, N., Bar-Mag, T., Lankry, D. and Mandelboim, O. (2012). MiR-10b downregulates the stress-induced cell surface molecule MICB, a critical ligand for cancer cell recognition by natural killer cells. Cancer Res 72(21): 5463-5472.

简介

肺清除测定测试先天免疫细胞(主要是NK细胞)根除通过小鼠尾静脉注射的体内细胞的能力。 该测定有助于阐明NK细胞及其受体(如果小鼠被遗传修饰)在体内的各种人和小鼠靶标上发挥的作用(Stern-Ginossar等人, ,2008; Halfteck等人,2009; Tsukerman等人,2012)。

关键字:NK, 在Vivo杀害, 肺清除

材料和试剂

  1. 细胞标记染料
    1. Vybrant CM-DiI(Life Technologies,Invitrogen TM,目录号:V-22888)
    2. VybrantDiD(Life Technologies,Invitrogen TM ,目录号:V-22887)
    3. 基于优选的波长,可以使用CellTrace CFSE(Life Technologies,Invitrogen TM ,目录号:C-34554)(CF-1或FL-2的优选波长) CM-DiI)
  2. 其他材料
    1. 磷酸盐缓冲盐水(PBS)
    2. 1×Dulbecco改良的Eagle培养基(DMEM)(Invitrogen,Life Technologies,Invitrogen TM ,目录号:10313-039)
    3. 胎牛血清(Life Technologies,Invitrogen TM ,目录号:11091-148)
    4. 40微米细胞滤器(BD Biosciences,Falcon ,目录号:352340)
    5. 感兴趣的小鼠
    6. NaN 3(叠氮化钠)
    7. 红细胞裂解缓冲液(ELB)(参见配方)
    8. FACS缓冲区(参见配方)
  3. 抗体
    为了证明观察到的效果是特异的,我们推荐阻断相关受体,或通过耗尽NK细胞,通过使用抗NK1.1(在受体存在的情况下),或通过使用抗脱唾液酸GM1。
    1. 抗NK1.1(eBioscience,目录号:14-5941-82)
    2. 抗-Asialo-GM1(eBioscience,目录号:16-6507-39)
    3. 抗小鼠CD314(NKG2D)(eBioscience,目录号:14-5873-82)

设备

  1. BD LSR II流式细胞仪

程序

  1. 尾静脉注射需要事先实践,成熟的雄性小鼠更容易注射。我们建议使用8-12周的雄性小鼠
  2. 在实验前通过I.P(腹膜内)注射150微克(在高达250μlPBS中)抗NK1.1至24小时,获得NK细胞耗竭一天的时间。
  3. 注意:不是所有的细胞类型都适合这些实验,小细胞很难恢复,大细胞可能堵塞肺,一些细胞没有标记好等。因此,强烈建议使用有限数量的动物的初步实验。我们测试的线是:HeLa,PC-3,DU145,PD1.6和YAC-1。由于尾静脉注射可在不同的小鼠之间变化,即使在同一组中,需要内部注射对照,其通常是对小鼠NK细胞杀伤有抗性的细胞。根据制造商的说明为染料标记两种细胞类型(实验和对照)。如果使用的细胞不在制造商方案中,则需要校准体外染色实验。如下进行HeLa(对于我们的大多数实验的对照群体),Jurkat,3T3,P3X,PC-3 DU145的标记:
    1. 将细胞以1×10 6个/ml的密度悬浮于无血清培养基(DMEM或RPMI)中。
    2. 加入1-5μl(从库存)的细胞标记溶液/ml细胞悬浮液。轻轻吹打混匀。在每个标记管中不要超过5ml体积。 CFSE和Vybrant-CM-DiI之间的差异是发射(FL-1/FL-2)和标记细胞的范围(我们建议在可能的情况下使用Vybrant-CM-DiI)。
    3. 在37℃HeLa(8分钟),Jurkat(2分钟),3T3(15分钟),P3X(15分钟),PC-3(30分钟)DU145(30分钟)孵育。 我们建议在用铝箔覆盖的15ml管中孵育
    4. 以450-515em g离心5分钟。
    5. 取出上清液,轻轻地在温热(37℃)培养基中重悬细胞
    6. 重复洗涤程序(3-d和3-e)。
  4. 将细胞重悬于终浓度为10 7个细胞/ml的这些细胞注射到小鼠,每种细胞类型的2×10 6个细胞(4×10 6个细胞/ 6总共)通过尾静脉注射每只小鼠; 最终注射体积为400μl。
  5. 将细胞通过一个40微米的细胞过滤器
  6. 留出400微升的细胞混合物,将作为预注射管。 在实验期间将这些细胞保持在37℃。
  7. 将细胞注入小鼠的尾静脉。
  8. 等待5小时。
  9. 根据伦理规定牺牲小鼠,我们建议使用CO 。
  10. 收获肺,将其放置在6孔板中,用2ml冰冷的PBS,直到完全收获整个实验的肺。
  11. 在50 ml管的顶部放置一个40μm细胞过滤器,将肺转移到过滤器中,将PBS从6孔转移到管中。
  12. 使用5ml注射器的圆筒/杵粉碎肺; 根据需要添加更多PBS。
  13. 在获得均匀溶液后,将管在450-515×g下离心5分钟。
  14. 弃去上清液并用10ml ELB(红细胞裂解缓冲液)重悬细胞并保持在冰上5分钟。
  15. 在450-515×g离心细胞5分钟。
  16. 弃去上清液并观察红细胞含量(如果沉淀中有可见的红细胞)重复步骤15-16,直到红细胞消失。
  17. 将细胞重悬在10ml补充有10%FBS的DMEM/RPMI中(该步骤消除ELB活性)。
  18. 离心细胞在450-515×g 5分钟。
  19. 弃去上清液和悬浮细胞在1-2毫升FACS缓冲区根据topellet大小。
  20. 使用FACS检查细胞。从预注样品开始,基于正向和侧向散射设置门(参见下图),并校准激光强度。收集至少2.5×10 5个细胞。绘制FL-4与FL-2(如果使用DiI)或FL-4与FL-1(如果使用CFSE)的点图。
  21. 阅读肺样本。我们建议每个样品至少收集2.5 x 10 6个细胞
  22. 注意:细胞可以重悬在2%甲醛中,并在4℃下在黑暗中储存过夜,然后通过流式细胞术进行分析。
  23. 流式细胞术分析(图1):Y轴FL-1强度X轴FL-4强度。方框标记"实验"细胞群;虚线方框标记"内部对照"细胞群体。左下方是小鼠的肺细胞(FL-1,FL-4阴性)。


    图1.流式细胞术分析

  24. 为了获得杀死百分比,计算预注射组中的实验细胞的量(NK灵敏的)除以对照细胞的量(NK不敏感的),并且将该比率设置为参考对照。 因此,对于感染后5小时在肺中回收的细胞进行类似的计算,并计算肿瘤细胞存活。

食谱

  1. ELB(红细胞裂解缓冲液)
    NH 4 Cl 16.4g
    KHCO 3 2 g
    EDTA 0.5M400μl
    2 L ddH 2 O 2/b 用HCl滴定至pH 7.2-7.4
  2. FACS缓冲区
    1x PBS
    0.5%BSA
    0.05%NaN 3(叠氮化钠)

致谢

作者要感谢Noam Stern-Ginossar,Chamutal Gur,Ariella Glasner和Mazal El-Nekaveh。

参考文献

  1. Halfteck,G.G.,Elboim,M.,Gur,C.,Achdout,H.,Ghadially,H.and Mandelboim,O.(2009)。 增强体内淋巴瘤肿瘤的生长在缺乏NK-激活受体NKp46/NCR1。 J Immunol 182(4):2221-2230。
  2. Stern-Ginossar,N.,Gur,C.,Biton,M.,Horwitz,E.,Elboim,M.,Stanietsky,N.,Mandelboim,M.and Mandelboim,O.(2008)。 人类microRNA调节受体NKG2D介导的应激诱导免疫反应。 Nat Immunol 9(9):1065-1073。
  3. Tsukerman,P.,Stern-Ginossar,N.,Gur,C.,Glasner,A.,Nachmani,D.,Bauman,Y.,Yamin,R.,Vitenshtein,A.,Stanietsky,N.,Bar-Mag ,T.,Lankry,D。和Mandelboim,O。(2012)。 MiR-10b下调应激诱导的细胞表面分子MICB,是癌细胞识别的关键配体自然杀伤细胞。 Cancer Res 72(21):5463-5472。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tsukerman, P. and Mandelboim, O. (2013). Lung Clearance Assay. Bio-protocol 3(5): e336. DOI: 10.21769/BioProtoc.336.
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