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
发布: 2015年06月20日第5卷第12期 DOI: 10.21769/BioProtoc.1501 浏览次数: 18169
评审: Thomas J. BartoshAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
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
- Alexa700-conjugated mouse anti-human CD7 clone 124-1D1 (eBioscience, catalog number: 56-0079-42 )
- 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 . - Phycoerythrin (PE)-Texas Red (ECD)-conjugated mouse anti-human CD3 clone UCHT1 (Beckman Coulter, catalog number: IM2705U )
- ECD-conjugated mouse anti-human CD14 clone RMO52 (Beckman Coulter, catalog number: IM2707U )
- PE-Cy7-conjugated mouse anti-human CD56 clone NCAM16.2 (BD Biosciences, catalog number: 335791 )
- Pacific Blue-conjugated mouse anti-human CD16 clone 3G8 (BD Biosciences, catalog number: 558122 )
- APC-Cy7-conjugated mouse anti-human CD19 clone SJ25C1 (BD Biosciences, catalog number: 557791 )
- Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD107a clone H4A3 (BD Biosciences, catalog number: 555800 )
- APC-conjugated mouse anti-human IFNγ clone B27 (BD Biosciences, catalog number: 554702 )
- Human IgG (Sigma-Aldrich, catalog number: I4506 )
- Anti–mouse immunoglobulin G–coated compensation beads (BD Biosciences, catalog number: 552843 )
- Amine Aqua Reactive Dye (AARD) (Life Technologies, catalog number: L34957 )
- 96 well U bottom plate (Corning, catalog number: 353077 )
- 96 well V bottom plate (Corning, catalog number: 3894 )
- RPMI (Life technologies, catalog number: 11875)
- L-Glutamine 200 mM (100x) (Life technologies, catalog number: 25030 )
- Penicillin (10,000 Units/ml)-Streptomycin (10,000 μg/ml) (Life technologies, catalog number: 15140 )
- Fetal bovine serum (Hyclone, catalog number: SH30071 )
- Buffy coats from Stanford Blood Center used to obtain Peripheral Blood Mononuclear Cells (PBMC)
- Ficoll-Paque Premium (GE Healthcare, catalog number: 17-5442-03 )
- Cryopreserved PBMC samples from San Francisco based HIV-1 infected cohorts SCOPE and OPTIONS
- Recombinant IL-12 (Peprotech, catalog number: 200-12 )
- Recombinant IL-18 (MBL & Biological Laboratories, catalog number: B001-5 )
- Brefeldin A from Penicilium brefeldianum (Sigma-Aldrich, catalog number: B7651 )
- BD golgi stop protein transport inhibitor containing monensin (BD Biosciences, catalog number: 554724 )
- Phosphate buffered saline (PBS) (Corning, catalog number: 21-040-CV )
- Ethylenediaminetetraacetic Acid (EDTA) (Teknova, catalog number: E0306 )
- Bovine Serum Albumin (BSA) (Gemini BioProducts, catalog number: 700-100P )
- 16% Paraformaldehyde (PFA) (Electron Microscopy Science, catalog number: 15710 )
- BD FACS Permeabilizing solution 2 (BD Biosciences, catalog number: 340973 )
- Cell Culture Grade Water (HyClone, catalog number: SH30529.02 )
- Deoxyribonuclease (DNase) I (Sigma-Aldrich, catalog number: DN25 )
- 15 ml conicals (Thermo Fisher Scientific, catalog number: 05-539-5 )
- Trypan blue in PBS (0.4% w/v) (Corning, catalog number: 25-900-CI )
- Complete media (see Recipes)
- FACS buffer (see Recipes)
- Paraformaldehyde recipe (see Recipes)
Equipment
- Biosafety cabinet (Nuaire, model: 407FM600 )
- 37 °C water bath (Cole Parmer)
- Calibrated single-channel and multi-channel pipettes
- Pipet-aid
- Tips (10 μl, 20 μl, 200 μl, 1,000 μl)
- Centrifuge (Beckman Coulter, Allegra 6R , rotor GH-3.8)
- 37° Celsius Incubator (Thermo Forma, model: 3110 )
- Nikon Optiphot microscope for counting cells
- Hemacytometer for counting cells (Hausser Scientific, catalog number: 1490 )
- 4-laser (405 nm, 488 nm, 532 nm and 633 nm) BD LSR-II
Software
- FlowJo Single Cell Analysis software
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
免疫学 > 免疫细胞功能 > 细胞毒性
免疫学 > 免疫细胞染色 > 流式细胞术
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