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Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining
人自然杀伤细胞(NK)的细胞体外刺激和 IFNγ 及CD107a 细胞因子胞内染色   

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Abstract

Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3neg, CD14neg, CD19neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with target cells or cytokines. This method can be used to assess the general functional capacity of NK cells in peripheral blood mononuclear cells from a wide range of study participants.

Keywords: NK cell(NK细胞), Natural Killer Cell(自然杀伤细胞), Degranulation(脱颗粒), Cytokine staining(细胞因子染色), CD7(CD7)

Materials and Reagents

  1. Alexa700-conjugated mouse anti-human CD7 clone 124-1D1 (eBioscience, catalog number: 56-0079-42 )
  2. K562 cell line (Kindly provided by Dr. Lewis L. Lanier, University of California, San Francisco, USA)
    Note: These can also be purchased from ATCC, catalog number CCL-243 .
  3. Phycoerythrin (PE)-Texas Red (ECD)-conjugated mouse anti-human CD3 clone UCHT1 (Beckman Coulter, catalog number: IM2705U )
  4. ECD-conjugated mouse anti-human CD14 clone RMO52 (Beckman Coulter, catalog number: IM2707U )
  5. PE-Cy7-conjugated mouse anti-human CD56 clone NCAM16.2 (BD Biosciences, catalog number: 335791 )
  6. Pacific Blue-conjugated mouse anti-human CD16 clone 3G8 (BD Biosciences, catalog number: 558122 )
  7. APC-Cy7-conjugated mouse anti-human CD19 clone SJ25C1 (BD Biosciences, catalog number: 557791 )
  8. Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD107a clone H4A3 (BD Biosciences, catalog number: 555800 )
  9. APC-conjugated mouse anti-human IFNγ clone B27 (BD Biosciences, catalog number: 554702 )
  10. Human IgG (Sigma-Aldrich, catalog number: I4506 )
  11. Anti–mouse immunoglobulin G–coated compensation beads (BD Biosciences, catalog number: 552843 )
  12. Amine Aqua Reactive Dye (AARD) (Life Technologies, catalog number: L34957 )
  13. 96 well U bottom plate (Corning, catalog number: 353077 )
  14. 96 well V bottom plate (Corning, catalog number: 3894 )
  15. RPMI (Life technologies, catalog number: 11875)
  16. L-Glutamine 200 mM (100x) (Life technologies, catalog number: 25030 )
  17. Penicillin (10,000 Units/ml)-Streptomycin (10,000 μg/ml) (Life technologies, catalog number: 15140 )
  18. Fetal bovine serum (Hyclone, catalog number: SH30071 )
  19. Buffy coats from Stanford Blood Center used to obtain Peripheral Blood Mononuclear Cells (PBMC)
  20. Ficoll-Paque Premium (GE Healthcare, catalog number: 17-5442-03 )
  21. Cryopreserved PBMC samples from San Francisco based HIV-1 infected cohorts SCOPE and OPTIONS
  22. Recombinant IL-12 (Peprotech, catalog number: 200-12 )
  23. Recombinant IL-18 (MBL & Biological Laboratories, catalog number: B001-5 )
  24. Brefeldin A from Penicilium brefeldianum (Sigma-Aldrich, catalog number: B7651 )
  25. BD golgi stop protein transport inhibitor containing monensin (BD Biosciences, catalog number: 554724 )
  26. Phosphate buffered saline (PBS) (Corning, catalog number: 21-040-CV )
  27. Ethylenediaminetetraacetic Acid (EDTA) (Teknova, catalog number: E0306 )
  28. Bovine Serum Albumin (BSA) (Gemini BioProducts, catalog number: 700-100P )
  29. 16% Paraformaldehyde (PFA) (Electron Microscopy Science, catalog number: 15710 )
  30. BD FACS Permeabilizing solution 2 (BD Biosciences, catalog number: 340973 )
  31. Cell Culture Grade Water (HyClone, catalog number: SH30529.02 )
  32. Deoxyribonuclease (DNase) I (Sigma-Aldrich, catalog number: DN25 )
  33. 15 ml conicals (Thermo Fisher Scientific, catalog number: 05-539-5 )
  34. Trypan blue in PBS (0.4% w/v) (Corning, catalog number: 25-900-CI )
  35. Complete media (see Recipes)
  36. FACS buffer (see Recipes)
  37. Paraformaldehyde recipe (see Recipes)

Equipment

  1. Biosafety cabinet (Nuaire, model: 407FM600 )
  2. 37 °C water bath (Cole Parmer)
  3. Calibrated single-channel and multi-channel pipettes
  4. Pipet-aid
  5. Tips (10 μl, 20 μl, 200 μl, 1,000 μl)
  6. Centrifuge (Beckman Coulter, Allegra 6R , rotor GH-3.8)
  7. 37° Celsius Incubator (Thermo Forma, model: 3110 )
  8. Nikon Optiphot microscope for counting cells
  9. Hemacytometer for counting cells (Hausser Scientific, catalog number: 1490 )
  10. 4-laser (405 nm, 488 nm, 532 nm and 633 nm) BD LSR-II

Software

  1. FlowJo Single Cell Analysis software

Procedure

  1. Recovery of Peripheral Blood Mononuclear Cells (PBMCs) from cryostorage.
    1. Warm complete media in a 37 °C water bath prior to thawing.
    2. Transfer frozen vials of cryopreserved PBMC from a cryofreezer into a 37 °C water bath and gently move the tube back and forth in the water, allowing the contents of the vial to thaw until there is only a small amount left frozen. Do not leave the cryovial unattended during the thawing process. Thawing will take approximately 1-2 min.
    3. In a biosafety cabinet, remove the liquid from the cryovial and place into a 15 ml conical tube using a 2 ml serological pipette and pipettor.
    4. Slowly add 10 ml of pre-warmed (37 °C) media dropwise into the 15 ml conical tube. Gently flick the tube as you are adding media to ensure that the liquid is well mixed.
    5. Cap the tube and centrifuge at 330 x g for 5 min in a pre-cooled 4 °C centrifuge.
    6. Decant supernatant, break up the pellet by flicking the tube, and resuspend in 10 ml of complete media containing DNase (0.01 mg/ml) to count cells.
    7. Mix 10 μl of cells with 10 μl of Trypan blue. Take 10 μl of that mixture and place onto a hemacytometer for counting.
    8. Centrifuge cells at 330 x g for 5 min at 4 °C, decant supernatant, break up the cell pellet by flicking the tube, and resuspend in complete media containing DNase at a concentration of 5 million cells per ml.
  2. Plate 500,000 PBMC per well in 100 μl by single channel pipette in a 96 well U bottom plate.
    Note: Alternative protocol for stimulation: Thawed PBMC can be incubated overnight (16-20 h) at 37 °C and 5% CO2 in complete media supplemented with 200 IU/ml human recombinant IL-2 (NCI BRB Preclinical Repository). This significantly increases the responsiveness of the NK cells to stimulation with target cells; however, culturing overnight in IL-2 can also alter the phenotype of the NK cells. One needs to consider what the desired readout for the experiment is in choosing between the main protocol and this alternative approach.
  3. Add FITC-conjugated anti-CD107a antibody using a single channel pipette at a dilution of 1:50 (i.e. 4 μl in 200 μl culture conditions) to all wells of a 96 well plate containing cells.
    Notes:
    1. The dilution of this antibody should be titrated for use with a particular cytometer.
    2. The final volume in each well should be 200 μl. There will be 100 μl of PBMC and either 100 μl of target cells (K562) or 100 μl of cytokine mixture.
  4. For target cell stimulation (i.e. K562), count and suspend 500,000 K562 cells in 1 ml of complete media containing DNase and add 50,000 target cells (100 μl) using a single channel pipette to appropriate wells.
  5. For cytokine stimulation, add IL-12 and IL-18 at a final concentration of 10 ng per cytokine per well. Add 1 μl of each cytokine to 998 μl complete media containing DNase. Add 100 μl per well using a single channel pipette to appropriate wells for stimulation.
  6. Mix all wells with a multichannel pipette by carefully pipetting up and down 3 times being careful not to tocause bubbles.
  7. Place plate in incubator for 1 h at 37 °C and 5% CO2.
  8. After 1 h, centrifuge plate at 330 x g in a 25 °C centrifuge.
  9. Remove 50 μl supernatant from each well being careful not to disturb cell pellet. To not disturb the pellet, tilt the plate at a 45-60° angle, and using a multichannel pipette, place tip to the side of the well and remove liquid slowly. The supernatant can be discarded.
  10. Prepare a master mix containing Brefeldin A (BFA) and GolgiStop (monensin) mixture.
    1. Dilute GolgiStop 7.8 μl into 42.2 μl complete media containing 0.01mg/ml DNase.
    2. Make a master mix of: 20 μl 10 mg/ml Brefeldin A (BFA)
      20 μl of diluted GolgiStop mixture from step 10a.
      4,960 μl complete media containing DNase
    3. Plate 50 μl master mix into each well using a single channel pipette. Mix well using a multichannel pipette.
  11. Incubate another 5 h at 37 °C, 5% CO2.
  12. Transfer cells to a 96 well V bottom plate. Centrifuge plate at 330 x g for 5 min at 4 °C and remove supernatant. Flick supernatant into a container to be discarded. To flick, turn the plate over and give one firm shake.
  13. Resuspend cells in 200 μl FACS buffer using a multichannel pipette and centrifuge plate at 330 x g for 5 min at 4 °C.
  14. Wash plate again adding 200 μl FACS buffer per well using a multichannel pipette. Centrifuge plate at 330 x g for 5 min at 4 °C. Flick supernatant into a container to be discarded. To stop the protocol overnight at this point, see step To continue, skip to step 16.
  15. For time considerations, the protocol can now be stopped here overnight. Resuspend cells in 200 μl FACS buffer using a multichannel pipette, place in the refrigerator overnight, and stain the next day. Prior to staining on Day 2, centrifuge plate at 330 x g for 5 min at 4 °C. Flick out supernatant. Blot the plate on paper towels. Then continue with step 16.
  16. Surface stain cells.
    1. Make up a master mixture of surface antibodies diluted in FACS buffer that have previously been titrated for your flow cytometer. Staining is performed in 50 μl volume for 500,000 cells.
      1. We have found the following combination works well for our BD LSR-II.

        Antigen
        Fluorophore
        Clone
        Dilutions
        CD3
        ECD
        UCHT1
        1:50
        CD14
        ECD
        RMO52
        1:400
        CD19
        APCCy7
        SJ25C1
        1:100
        CD7
        Alexa700
        124-1D1
        1:50
        CD16
        Pacific Blue
        3G8
        1:100
        CD56
        PECy7
        NCAM16.2
        1:100
        IgG (100 μg/mL) 1:10



        Live/Dead Marker
        Amine Aqua Reactive Dye (AARD)

        1:200
        FACS buffer




      2. The use of fluorescence minus one (FMOs) is recommended. These can be made using the exact same mixture of antibodies but omitting one antibody. We focus on FMOs for CD107a and IFNγ, although FMOs can be used for all fluorophores initially to set appropriate gating of cytometry data.
      3. Make sure to have centrifuged the plate at 330 x g for 5 min at 4 °C and flicked out the supernatant prior to adding the surface stain. Aliquot 50 μl of antibody cocktail into each well using a single channel pipette. Mix well by pipetting up and down using a multichannel pipette. Allow plate to incubate on ice for 30 min to stain.
  17. After 30 min, add 150 μl FACS buffer to all wells using a multichannel pipette, centrifuge plate at 515 x g for 5 min at 4 °C. To improve recovery of cells from this step forward, a faster centrifuge speed is used.
  18. Flick supernatant into a container to be discarded and blot the plate on paper towels.
  19. Fix cells by adding 100 μl per well of 2% paraformaldehyde in PBS on ice for 20 min using a multichannel pipette.
  20. Spin at 915 x g in a 4 °C centrifuge for 5 min.
  21. Flick supernatant into a container to be discarded and blot on paper towels.
  22. Permeabilize cells using BD FACS Permeabilizing solution 2.
    1. Perm2 is supplied as a 10x solution. Dilute to 1x using cell culture grade water.
    2. Add 75 μl of the 1x Perm2 solution per well using a multichannel pipette and pipet up and down to resuspend cell pellet.
    3. Incubate plate for 10 min only at room temperature.
      1. Shorter incubations will reduce intracellular staining while incubating longer than 10 min will significantly affect cellular integrity and result in loss of sample.
  23. Wash once by adding 125 μl FACS buffer to each well using a multichannel pipette.
  24. Centrifuge plate at 915 x g in a 4 °C centrifuge for 5 min.
  25. Flick supernatant into a container to be discarded and blot the plate on paper towels.
  26. Intracellularly stain cells.
    1. Make up an antibody master mix with APC-conjugated anti-IFNγ antibody diluted into FACS buffer (do not dilute into Perm2 buffer).
    2. Aliquot 50 μl of antibody cocktail into each well by single channel pipette. Mix well by pipetting up and down using a multichannel pipette. Allow plate to incubate on ice for 30 min to stain.
  27. Wash once by adding 150 μl FACS buffer to each well using a multichannel pipette. Centrifuge plate at 915 x g in a 4 °C centrifuge for 5 min.
  28. Flick supernatant into a container to be discarded and blot plate on paper towels.
  29. To fix, add 50 μl per well of 2% paraformaldehyde diluted with PBS using a multichannel pipette. Cells should be fixed a minimum of 10 min on ice, but samples can be stored at 4 °C for up to 2 days without significant changes in fluorescent staining. The fixative does not need to be washed out prior to analysis.
  30. Run cells on BD LSR-II.
  31. Data are analyzed using FlowJo Single Cell Analysis software. Gates for IFNγ and CD107a expression are set using FMOs on a control sample and applied to all other samples collected. An example of the gating strategy and results of IFNγ and CD107a expression following media only (i.e. no stimulation), K562 target cell stimulation and IL-12 + IL-18 stimulation are shown (Figure 1).


    Figure 1. Representative gating strategy to identify NK cells and responses to stimulation. NK cells are identified as live lymphocytes that are CD3, CD14 and CD19 negative, but express CD7, CD56 and CD16. K562 target cell stimulation induces expression of both CD107a and IFNγ. IL-12 + IL-18 cytokine stimulation induces a robust IFNγ response from NK cells, however these cytokines do not result in degranulation as measured by CD107a expression.

Recipes

  1. Complete media (make fresh each time)
    RPMI
    10% Fetal Bovine Serum
    2 mM L-glutamine
    Penicillin (100 Units/ml)/Streptomycin (100 μg/ml)
    DNase 0.01 mg/ml
  2. FACS buffer (shelf life 1 month)
    Phosphate buffer saline
    0.5% Bovine serum albumin
    2 mM Ethylenediaminetetraacetic Acid
  3. Paraformaldehyde (PFA) recipe
    Dilution of PFA made fresh each time
    Dilute 16% stock PFA in PBS to a final concentration of 2%

Acknowledgments

This protocol has been adapted from the publications by Milush et al. (2009 and 2013). This research was supported, in part, by the Department of Health and Human Services funding under NIH Grant number 5T32HL007185 to JMM.

References

  1. Milush, J. M., Lopez-Verges, S., York, V. A., Deeks, S. G., Martin, J. N., Hecht, F. M., Lanier, L. L. and Nixon, D. F. (2013). CD56negCD16(+) NK cells are activated mature NK cells with impaired effector function during HIV-1 infection. Retrovirology 10: 158.
  2. Milush, J. M., Long, B. R., Snyder-Cappione, J. E., Cappione, A. J., 3rd, York, V. A., Ndhlovu, L. C., Lanier, L. L., Michaelsson, J. and Nixon, D. F. (2009). Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4. Blood 114(23): 4823-4831.

简介

自然杀伤(NK)细胞在人中包含5-20%的外周血单核细胞(PBMC)。 除了它们在防御病毒感染和肿瘤监测中的基本作用,NK细胞通过其细胞因子的产生帮助形成适应性免疫应答。 NK细胞传统上被鉴定为表达CD56的CD3阴性,CD14阳性,CD19阴性淋巴细胞。 使用包括CD56和CD7的标记物的组合极大地增加了定义NK细胞亚群的表型和功能的能力。 NK细胞功能的两个关键标记是IFNγ的产生和通过CD107a的表达测量的细胞毒性颗粒的释放。 在这里我们描述了一种方法来评估在靶细胞或细胞因子刺激后NK细胞中的IFNγ和CD107a表达。 该方法可用于评估来自广泛的研究参与者的外周血单核细胞中NK细胞的一般功能能力。

关键字:NK细胞, 自然杀伤细胞, 脱颗粒, 细胞因子染色, CD7

材料和试剂

  1. Alexa700缀合的小鼠抗人CD7克隆124-1D1(eBioscience,目录号:56-0079-42)
  2. K562细胞系(由Lewis L.Lanier博士提供,加利福尼亚大学,旧金山,美国) 注意:这些也可以从ATCC购买,目录号为CCL-243。
  3. 藻红蛋白(PE) - 德克萨斯红(ECD) - 缀合的小鼠抗人CD3克隆UCHT1(Beckman Coulter,目录号:IM2705U)
  4. ECD-缀合的小鼠抗人CD14克隆RMO52(Beckman Coulter,目录号:IM2707U)
  5. PE-Cy7缀合的小鼠抗人CD56克隆NCAM16.2(BD Biosciences,目录号:335791)
  6. 太平洋蓝标记的小鼠抗人CD16克隆3G8(BD Biosciences,目录号:558122)
  7. APC-Cy7缀合的小鼠抗人CD19克隆SJ25C1(BD Biosciences,目录号:557791)
  8. 荧光素异硫氰酸酯(FITC)结合的小鼠抗人CD107a克隆H4A3(BD Biosciences,目录号:555800)
  9. APC缀合的小鼠抗人IFNγ克隆B27(BD Biosciences,目录号:554702)
  10. 人IgG(Sigma-Aldrich,目录号:I4506)
  11. 抗小鼠免疫球蛋白G包被的补偿珠(BD Biosciences,目录号:552843)
  12. 胺水反应性染料(AARD)(Life Technologies,目录号:L34957)
  13. 96孔U底板(Corning,目录号:353077)
  14. 96孔V底板(Corning,目录号:3894)
  15. RPMI(生命技术,目录号:11875)
  16. L-谷氨酰胺200mM(100x)(Life technologies,目录号:25030)
  17. 青霉素(10,000单位/ml) - 链霉素(10,000μg/ml)(Life technologies,目录号:15140)
  18. 胎牛血清(Hyclone,目录号:SH30071)
  19. 来自斯坦福血液中心的血沉棕黄层用于获得外周血单核细胞(PBMC)
  20. Ficoll-Paque Premium(GE Healthcare,目录号:17-5442-03)
  21. 来自旧金山的HIV-1感染的队列范围和选项的冷冻保存的PBMC样品
  22. 重组IL-12(Peprotech,目录号:200-12)
  23. 重组IL-18(MBL& Biological Laboratories,目录号:B001-5)
  24. 来自布鲁氏菌Penicilium brefeldianum的Brefeldin A(Sigma-Aldrich,目录号:B7651)
  25. BD高尔基体蛋白转运抑制剂含有莫能菌素(BD Biosciences,目录号:554724)
  26. 磷酸盐缓冲盐水(PBS)(Corning,目录号:21-040-CV)
  27. 乙二胺四乙酸(EDTA)(Teknova,目录号:E0306)
  28. 牛血清白蛋白(BSA)(Gemini BioProducts,目录号:700-100P)
  29. 16%多聚甲醛(PFA)(Electron Microscopy Science,目录号:15710)
  30. BD FACS Permeabilizing solution 2(BD Biosciences,目录号:340973)
  31. 细胞培养级水(HyClone,目录号:SH30529.02)
  32. 脱氧核糖核酸酶(DNase)I(Sigma-Aldrich,目录号:DN25)
  33. 15ml锥形瓶(Thermo Fisher Scientific,目录号:05-539-5)
  34. PBS(0.4%w/v)(Corning,目录号:25-900-CI)中的台盼蓝
  35. 完成媒体(见配方)
  36. FACS缓冲区(参见配方)
  37. 多聚甲醛食谱(见配方)

设备

  1. 生物安全柜(Nuaire,型号:407FM600)
  2. 37℃水浴(Cole Parmer)
  3. 校准的单通道和多通道移液器
  4. 助洗剂
  5. 提示(10μl,20μl,200μl,1,000μl)
  6. 离心机(Beckman Coulter,Allegra 6R,转子GH-3.8)
  7. 37℃孵育器(Thermo Forma,型号:3110)
  8. 用于计数细胞的尼康Optiphot显微镜
  9. 用于计数细胞的血细胞计数器(Hausser Scientific,目录号:1490)
  10. 4激光(405nm,488nm,532nm和633nm)BD LSR-II

软件

  1. FlowJo单细胞分析软件

程序

  1. 从冷冻保存中回收外周血单核细胞(PBMC)
    1. 在解冻前在37℃水浴中加热完全培养基。
    2. 将冷冻小瓶的冷冻保存的PBMC从冷冻冻存器转移到37℃   °C水浴并轻轻地在水中来回移动管, 允许小瓶的内容物解冻,直到只有小的 剩余冻结量。 不要离开冷冻无人看管 解冻过程。 解冻约需1-2分钟。
    3. 在一个 生物安全柜,从冷冻管中取出液体并放入 15ml锥形管,使用2ml血清移液管和移液器
    4. 缓慢加入10ml预热(37℃)培养基至15ml 锥形管。 在添加介质时轻轻弹动管,以确保 液体混合良好。
    5. 盖上管并在预冷的4℃离心机中以330×g离心5分钟。
    6. 倾析上清液,通过轻拂管破碎沉淀物,和 重悬于10ml含有DNase(0.01mg/ml)的完全培养基中 计数单元格
    7. 混合10微升细胞与10微升台盼蓝。 取10μl的混合物,放在血细胞计数器上计数。
    8. 在4℃下以330×g离心细胞5分钟,倾析上清液, 通过轻拂管破碎细胞沉淀,并重悬在完全   含有浓度为5百万个细胞/ml的DNA酶的培养基。
  2. 在96孔U底板中通过单通道移液管在100μl板中每孔500,000个PBMC 注释:用于刺激的替代方案:解冻的PBMC可以在37℃和5%CO 2下孵育过夜(16-20小时)。 补充有200IU/ml人重组IL-2(NCI BRB Preclinical 存储库)。这显着增加NK细胞对靶细胞刺激的反应性;然而,在IL-2中培养过夜也可以改变NK细胞的表型。需要考虑实验的期望读数在主协议和这种替代方法之间的选择。
  3. 使用单通道移液管以1:50的稀释度(即在200μl培养条件中的4μl)将FITC缀合的抗CD107a抗体加入含有细胞的96孔板的所有孔。
    注意:
    1. 此抗体的稀释度应与特定的血细胞计数器一起使用。
    2. 每个孔中的最终体积应为200μl。将有100微升  的PBMC和100μl靶细胞(K562)或100μl细胞因子 混合物。
  4. 对于靶细胞刺激(即,K562),计数并悬浮500,000个K562细胞于1ml含有DNase的完全培养基中,并使用单通道移液管加入50,000个靶细胞(100μl) 井
  5. 对于细胞因子刺激,以每孔细胞因子10ng的终浓度添加IL-12和IL-18。 加入1微升的每种细胞因子到998微升含有DNA酶的完全培养基中。 使用单通道移液器加入100微升/孔到适当的井进行刺激。
  6. 使用多通道移液器小心吸取上下3次,小心不要使气泡混浊,以混合所有孔。
  7. 将板在孵育器中在37℃和5%CO 2下孵育1小时
  8. 1小时后,在25℃离心机中以330×g离心板离心
  9. 从每个孔中删除50μl上清,小心不要打扰细胞沉淀。 为了不干扰沉淀,以45-60°的角度倾斜板,并使用多通道移液器,将尖端放置在孔的侧面,并缓慢地移除液体。 可以弃去上清液。
  10. 准备含有布雷菲德菌素A(BFA)和GolgiStop(莫能菌素)混合物的主混合物。
    1. 稀释GolgiStop 7.8μl到42.2μl含0.01mg/ml DNA酶的完整培养基
    2. 做一个主混合:20微升10毫克/毫升布雷菲德菌素A(BFA)
      20μl来自步骤10a的稀释的GolgiStop混合物 4,960μl含DNase的完整培养基
    3. 使用单通道移液器板50μl主混合物到每个孔。 使用多通道移液器混合好。
  11. 在37℃,5%CO 2下再孵育5小时
  12. 转移细胞到96孔V底板。在4℃下以330×g离心板5分钟,除去上清液。将上清液倒入容器中弃去。要轻拂,把盘子翻过来,给一个坚定的摇动
  13. 使用多通道移液管和离心机在330℃下在4℃下将细胞重悬于200μlFACS缓冲液中5分钟。
  14. 再次使用多通道移液器,每孔加入200μlFACS缓冲液洗涤板。在4℃下以330xg离心板离心5分钟。将上清液倒入容器中弃去。要在此时停止协议,请参阅步骤要继续,请跳至步骤16.
  15. 出于时间考虑,该协议现在可以在这里停止一夜。使用多通道移液器在200μlFACS缓冲液中重悬细胞,放在冰箱过夜,第二天染色。在第2天染色之前,在4℃下以330×g离心板离心5分钟。甩出上清液。将纸板上的纸巾。然后继续执行步骤16.
  16. 表面染色细胞。
    1. 组成在FACS缓冲液中稀释的表面抗体的主混合物 以前已为您的流式细胞仪滴定。 染色是 在50μl体积中进行500,000个细胞
      1. 我们发现以下组合适用于我们的BD LSR-II。

        抗原
        荧光团
        克隆
        稀释
        CD3
        ECD
        UCHT1
        1:50
        CD14
        ECD
        RMO52
        1:400
        CD19
        APCCy7
        SJ25C1
        1:100
        CD7
        Alexa700
        124-1D1
        1:50
        CD16
        太平洋蓝
        3G8
        1:100
        CD56
        PECy7
        NCAM16.2
        1:100
        IgG(100μg/mL)1:10



        活/死标记
        胺水反应性染料(AARD)

        1:200
        FACS缓冲区




      2. 建议使用荧光减1(FMOs)。 这些可以 使用完全相同的抗体混合物,但省略一个 抗体。 我们关注CD107a和IFNγ的FMO,尽管FMOs可以 用于所有荧光团最初设置适当的门控 细胞计数数据
      3. 确保在330 x离心板   在4℃下孵育5分钟,并在加入前将上清液轻轻倒出   表面污渍。 使用a。等分50μl抗体混合物到每个孔中   单通道移液器。 通过使用a。上下吹吸混匀 多通道移液器。 使板在冰上孵育30分钟 弄脏。
  17. 30分钟后,使用多通道移液管,在4℃下以515×g离心5分钟,向所有孔中加入150μlFACS缓冲液。 为了提高细胞从这一步的回收率,使用更快的离心机速度
  18. 将上清液倒入容器中弃去,并将该板在纸巾上吸干
  19. 通过使用多通道移液器在冰上加入100μl每孔的2%多聚甲醛的PBS溶液20分钟来修复细胞。
  20. 在4℃离心机中旋转5分钟,915×g下旋转
  21. 将上清液倒入容器中,弃去并在纸巾上打印
  22. 使用BD FACS Permeabilizing溶液2使细胞透化。
    1. Perm2作为10x解决方案提供。 用细胞培养级稀释至1倍。
    2. 每孔加入75μl的1x Perm2溶液,使用多通道移液器和吸移管上下重悬细胞沉淀。
    3. 仅在室温下孵育平板10分钟。
      1. 较短的孵育会减少细胞内染色 孵育超过10分钟将显着影响细胞 完整性并导致样品损失。
  23. 使用多通道移液器向每个孔中加入125μlFACS缓冲液洗涤一次
  24. 在4℃离心机中将板以915×g离心5分钟。
  25. 将上清液倒入容器中弃去,并将该板在纸巾上吸干
  26. 细胞内染色细胞。
    1. 用APC-偶联的抗-IFNγ抗体组成抗体主混合物 稀释到FACS缓冲液中(不稀释到Perm2缓冲液中)。
    2. 通过单通道等分50μl抗体混合物到每个孔 吸管。 使用多通道移液器通过上下吹吸混匀。 使板在冰上孵育30分钟以染色。
  27. 使用多通道移液器向每个孔中加入150μlFACS缓冲液洗涤一次。 在4℃离心机中将板以915×g离心5分钟。
  28. 将上清液倒入容器中弃去,并在纸巾上擦拭
  29. 要固定,使用多通道移液器加入50μl每孔的2%多聚甲醛用PBS稀释。 细胞应在冰上固定至少10分钟,但样品可在4℃下储存长达2天,而荧光染色没有显着变化。 固定剂在分析之前不需要洗去。
  30. 在BD LSR-II上运行细胞。
  31. 使用FlowJo单细胞分析软件分析数据。 使用对照样品上的FMO设置IFNγ和CD107a表达的门,并应用于收集的所有其他样品。 门控策略的一个例子 显示仅在培养基(即没有刺激),K562靶细胞刺激和IL-12 + IL-18刺激后的IFNγ和CD107a表达的结果(图1)。


    图1.鉴定NK细胞和刺激反应的代表性门控策略。NK细胞被鉴定为CD3,CD14和CD19阴性的活淋巴细胞,但表达CD7,CD56和CD16。 K562靶细胞刺激诱导CD107a和IFNγ二者的表达。 IL-12 + IL-18细胞因子刺激诱导来自NK细胞的强大的IFNγ应答,然而这些细胞因子不导致如通过CD107a表达测量的脱颗粒。

食谱

  1. 完整的媒体(每次新鲜)
    RPMI
    10%胎牛血清
    2mM L-谷氨酰胺 青霉素(100单位/ml)/链霉素(100μg/ml) DNase 0.01mg/ml
  2. FACS缓冲液(保质期1个月)
    磷酸盐缓冲盐水
    0.5%牛血清白蛋白
    2mM乙二胺四乙酸
  3. 多聚甲醛(PFA)配方
    每次稀释的PFA都是新鲜的
    在PBS中稀释16%储备PFA至最终浓度为2%

致谢

该协议已经由Milush等人的出版物(2009和2013)改编。 这项研究得到卫生和人类服务部资助的NIH资助,批准号为5T32HL007185给JMM。

参考文献

  1. Milush,J.M.,Lopez-Verges,S.,York,V.A.,Deeks,S.G.,Martin,J.N.,Hecht,F.M.,Lanier,L.L.and Nixon,D.F。 CD56negCD16(+)NK细胞在HIV-1感染期间被活化的具有受损的效应子功能的成熟NK细胞。 Retrovirology 10:158
  2. Milush,J.M.,Long,B.R.,Snyder-Cappione,J.E.,Cappione,A.J.,3rd,York,V.A.,Ndhlovu,L.C.,Lanier,L.L.,Michaelsson,J.and Nixon,D.F。 通过共表达CD56,CD7而鉴定的人NK细胞和单核细胞/DC样细胞的功能不同的亚群 ,和CD4。血液 114(23):4823-4831。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:York, V. A. and Milush, J. M. (2015). Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining. Bio-protocol 5(12): e1501. DOI: 10.21769/BioProtoc.1501.
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