Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells
用基于PLA原理的 Duolink®体系观察T淋巴细胞中蛋白相互作用的方法   

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Nature Immunology
Jul 2015



Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and solve this kind of issue. Moreover PLA experiments are read out using fluorescence or bright field microscopy, providing additional information on intracellular interactions localization significantly bettering classical IP procedures.

PLA reagents are made of complementary small oligonucleotides “minus” and “plus” probes which specifically recognize host species from the primary antibodies (Abs) targeting the two proteins you are interested in. Experiments have to be designed with primary Abs from different species (rabbit, mouse or goat) as PLA probes “minus” or “plus” react against a specific host species of the primary antibody (e.g. “plus” anti-rabbit with “minus” anti-mouse or “plus” anti-mouse with “minus” anti-rabbit combos are allowed if mouse and rabbit primary Abs are used). When the two PLA probes are close enough (40 nm) a ligation occurs upon ligase incubation generating a circle DNA. These circle-forming DNAs are next amplified thanks to a polymerase and complementary fluorescent nucleotides, being incorporated at this step. Each luminescent spot is thereafter considered being an interaction site between the two proteins (Figure 1).

Figure 1. Schematic representation of Duolink® experiment. Primary Abs from different host species were used [i.e., Mouse (Ms) anti-Nlrp3 and Rabbit (Rb) anti-IRF4]. When protein-protein interaction occurs PLA probes allow incorporation of fluorescent oligonucleotides which are analyzed by microscopy.

PLA experiments have been performed on differentiated T cells from mice. T cells were grown on cover slip coated with poly-L-Lysine. Please be aware that for T cells and other non-adherent cells, experiments and staining are also possible in 500 µl microtubes until the last washing step before mounting cover slip on slide. This microtube method saves cytokines reagents and allows T cells to grow with usual methods but centrifugation repetition for washing steps is hazardous. Primary Abs incubation also requires also a specific setup if microtube method is chosen.

Keywords: Protein interaction (蛋白质相互作用), Fluorescence (荧光), CD4 T cells (CD4 T细胞)

Materials and Reagents

  1. Glass cover slip (Ø13 mm thickness, No. 1, 5) (VWR International, catalog number: 631-0150 )
  2. Primary antibodies (Abs)
    1. Mouse anti-Nlrp3 (1/100) (Adipogen International, catalog number: AG-20B-0014 )
      Note: Starting concentration of mouse anti-Nlrp3 is1 µg/µl.
    2. Rabbit anri-IRF4 (1/100) (Novus Biologicals, catalog number: NBP1-00893 )
      Note: Starting concentration of rabbit anri-IRF4 is 0.08 µg/µl.
  3. Duolink® reagents
    1. Anti-rabbit PLUS probe (1/5) (Sigma-Aldrich, catalog number: Duo92002 )
    2. Anti-mouse MINUS probe (1/5) (Sigma-Aldrich, catalog number: Duo92004 )
    3. Ligation reagents (5x) (Sigma-Aldrich, catalog number: Duo92007 )
      Note: Ligation reagents, ligase and amplification reagents are all included in the pack # DUO92007 .
    4. Ligase (1 unit/μl) (Sigma-Aldrich, catalog number: Duo82029 )
    5. Amplification reagents (containing orange labeled oligonucleotides) (5x) (Sigma-Aldrich, catalog number: Duo92007 )
    6. Polymerase (10 unit/μl) (Sigma-Aldrich, catalog number: Duo82030 )
    7. Wash buffer A (Sigma-Aldrich, catalog number: Duo82049 )
    8. Wash buffer B (Sigma-Aldrich, catalog number: Duo82049 )
  4. Poly-L-Lysine (Sigma-Aldrich, catalog number: P4707 )
  5. Triton X-100 (0.1% in PBS) (Sigma-Aldrich, catalog number: T8787 )
  6. Blocking & Abs dilution buffer (0.5% BSA in PBS)
  7. Mounting medium containing DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36931 )
  8. 1x PBS (Lonza, catalog number: 17-516F )
  9. RNase free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977035 )
  10. 4% formaldehyde (PFA) (VWR International, catalog number: 9713.1000 )


  1. Pipets (from 1 to 1,000 µl)
  2. Fluorescence microscope (with DAPI and Cy2 or Cy3 emission filter) (ZEISS, model: Imager M2)
    Note: Objective 63x is recommended for T cells imaging.
  3. 37 °C incubator
  4. Freeze block for enzymes
  5. Orbital shaker


  1. ImageJ


  1. Day 0: Th2 and naïve T cells were isolated and differentiated as previously described (Bruchard et al., 2015). Approximately 107 cells were then transferred to 12-wells dishes containing a cover slip coated with a room temperature 10 min treatment with a 100-150 µl drop of poly-L-Lysine. Lysine was then removed by pipetting and wells were washed with 1 ml PBS. Cells were then seeded and allowed to attach to cover slip for 24 h at 37 °C.
  2. Day 1: Please note that all procedures from 1 to 5 are performed in the same 12-wells dishes for cell culture. T cells were briefly washed 2 times in 1 ml PBS by removing medium with a pipette and replacing with fresh PBS. Please be aware that cover slip shall never dry.
  3. T cells’ fixation was performed using 200 µl 4% PFA during 10 min at 4 °C followed by 2 washes of 1 ml PBS 1x in the well containing the cover slip.
  4. Permeabilization was obtained using 200 µl 0.1% Triton X-100 during 2 min at RT followed by 2 washes of 1 ml 1x PBS.
  5. Cover slips were blocked using 1 ml 0.5% BSA in PBS during 20 min at RT.
  6. Cover slips were incubated in a 30 to 40 µl drop of diluted primary antibodies (Abs) overnight at 4 °C (Figure 2).

    Figure 2. Cover slip handling for PLA procedures. A. A 60 mm diameter plastic petri dish was used as humidity chamber for Abs incubation and PLA experiments. Humidity was obtained with a 15 ml cap filled with water. Flat tip forceps were used to handle cover slips all along procedure. B. After being removed from culture dish, cover slips (*) were incubated face down on a 30 µl drop of Abs (†). C. Cover slip (*) placed upon the drop. From this step, the petri dish was closed and sealed with parafilm and placed overnight at 4 °C. D. The following day, cover slips were carefully picked up from the humidity chamber using forceps and a needle. Cover slips were then individually placed face up for washing steps in a well of a 6-wells plate filled with 3 ml of 1x PBS.
    Note: This procedure was also repeated for incubation of Duolink® products. It enabled to save Duolink® reagents compared to manufacturer’s instructions. All other manufacturer’s instructions (incubation times, washing steps and buffers...) were carefully followed.

  7. Day 2: Cover slips were washed 3 times 5 min in 1x PBS at RT.
  8. PLA experiment was performed using manufacturer’s instructions. The procedure is also available on bio-protocol website (Lin et al., 2015) until step 9.
  9. All samples were allowed to dry in the dark at RT.
  10. A drop of mounting medium containing DAPI was placed between each samples and slide. Slides were kept at 4 °C for short term storage.
  11. Day 3: Imaging procedures were performed using a fluorescence microscope with 63x objective equipped with appropriate filters (DAPI: Ex 365, Em: BP 445/50; Duolink spots: Ex: BP 550/25, Em: 605/70; Figure 3). Spots inside nuclei (as DAPI as the reference) were quantified using Image J and its Analyse particles tab. Slides were then kept at -20 °C for long term storage.

Representative data

Figure 3. Reprensative images for Duolink spots in TH2 cells. T cells pictures obtained with our protocol (Nv: Naïve, Scale bar in yellow: 5 µm). Red dots in Th2 cells are Duolink spots.


  1. Use freshly prepared BSA in PBS anytime as possible or filter the preparation if using an old solution. If this step is not taken into account, sediments could appear and interfere with spots detection.


This work was supported by a French Government grant managed by the French National Research Agency under the program “Investissements d’Avenir” with reference ANR-11-LABX-0021 (Lipstic Labex). FG team is « Equipe labélisée Ligue Nationale Contre le Cancer ». V. D. is the recipient of a « poste d’accueil INSERM »; This protocol was adapted from previous work of Fredriksson et al. (2007) and Lin et al. (2015).


  1. Bruchard, M., Rebe, C., Derangere, V., Togbe, D., Ryffel, B., Boidot, R., Humblin, E., Hamman, A., Chalmin, F., Berger, H., Chevriaux, A., Limagne, E., Apetoh, L., Vegran, F. and Ghiringhelli, F. (2015). The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nat Immunol 16(8): 859-870.
  2. Fredriksson, S., Dixon, W., Ji, H., Koong, A. C., Mindrinos, M. and Davis, R. W. (2007). Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat Methods 4(4): 327-329.
  3. Lin, M. Z., Martin, J. L. and Baxter, R. C. (2015). Proximity ligation assay (PLA) to detect protein-protein interactions in breast cancer. Bio-protocol 5(10): e1479.


蛋白质 - 蛋白质相互作用实验,例如共免疫沉淀(IP)测定,经典地需要大量的细胞。当您的工作关注稀有细胞群体(例如淋巴细胞亚型)时,这成为一个问题。 O-link Bioscience开发了邻近连接测定(PLA)试剂和程序以缓解和解决这类问题。此外,使用荧光或明视场显微镜读出PLA实验,提供关于细胞内相互作用定位的额外信息,显着改善经典IP程序。 PLA试剂由互补的小寡核苷酸"负"和"正"探针组成,其特异性识别来自靶向您感兴趣的两种蛋白质的一抗(Abs)的宿主物种。实验必须使用来自不同物种,小鼠或山羊)作为PLA探针"减"或"加"对一抗的特定宿主物种(例如,"加"抗兔与"减"抗鼠或"加" "如果使用小鼠和兔初级Abs,则允许使用"减"抗兔组合的抗小鼠)。当两种PLA探针足够接近(40nm)时,在连接酶孵育后发生连接,产生环DNA。接下来,由于在该步骤中掺入的聚合酶和互补荧光核苷酸,这些形成环的DNA被扩增。每个发光点此后被认为是两种蛋白质之间的相互作用位点(图1)。

图1. Duolink ®实验的示意图。 小鼠(Ms)抗-Nlrp3和兔(Rb)抗-IRF4]。当发生蛋白质 - 蛋白质相互作用时,PLA探针允许掺入通过显微镜分析的荧光寡核苷酸。

  对来自小鼠的分化的T细胞进行PLA实验。 T细胞在用聚-L-赖氨酸包被的盖玻片上生长。请注意,对于T细胞和其他非粘附细胞,实验和染色也可能在500μl微量管中,直到最后一次清洗步骤,然后将盖玻片安装在载玻片上。这种微管方法节省细胞因子试剂并允许T细胞用常规方法生长,但是洗涤步骤的重复离心是危险的。如果选择微管方法,主抗体孵育也需要特定的设置。

关键字:蛋白质相互作用, 荧光, CD4 T细胞


  1. 玻璃盖玻片(Ø13mm厚,1号,5号)(VWR国际,目录号:631-0150)
  2. 一级抗体(Abs)
    1. 小鼠抗Nlrp3(1/100)(Adipogen International,目录号:AG-20B-0014)
    2. 兔anri-IRF4(1/100)(Novus Biologicals,目录号:NBP1-00893)
  3. Duolink ®试剂
    1. 抗兔PLUS探针(1/5)(Sigma-Aldrich,目录号:Duo92002)
    2. 抗小鼠MINUS探针(1/5)(Sigma-Aldrich,目录号:Duo92004)
    3. 连接试剂(5x)(Sigma-Aldrich,目录号:Duo92007)
    4. 连接酶(1单位/μl)(Sigma-Aldrich,目录号:Duo82029)
    5. 扩增试剂(含有橙色标记的寡核苷酸)(5x)(Sigma-Aldrich,目录号:Duo92007)
    6. 聚合酶(10单位/μl)(Sigma-Aldrich,目录号:Duo82030)
    7. 洗涤缓冲液A(Sigma-Aldrich,目录号:Duo82049)
    8. 洗涤缓冲液B(Sigma-Aldrich,目录号:Duo82049)
  4. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)
  5. Triton X-100(0.1%在PBS中)(Sigma-Aldrich,目录号:T8787)
  6. 封锁& Abs稀释缓冲液(0.5%BSA的PBS溶液)
  7. 含有DAPI(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36931)的固定介质
  8. 1x PBS(Lonza,目录号:17-516F)
  9. 无RNA酶的水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977035)
  10. 4%甲醛(PFA)(VWR International,目录号:9713.1000)


  1. 移液管(1至1000μl)
  2. 荧光显微镜(使用DAPI和Cy2或Cy3发射滤光片)(ZEISS,型号:Imager M2)
  3. 37℃孵育器
  4. 酶冻结块
  5. 轨道振动器


  1. 图像J


  1. 第0天:如先前所述分离和分化Th2和初始T细胞(Bruchard等人,2015)。 然后将约10 7个细胞转移到含有盖玻片的12孔皿中,所述盖玻片用100-150μl的聚-L-赖氨酸滴室温处理10分钟。 然后通过移液除去赖氨酸,并用1ml PBS洗涤孔。 然后将细胞接种并使其在37℃下附着于盖玻片24小时
  2. 第1天:请注意,所有程序从1到5在相同的12孔培养皿中进行细胞培养。将T细胞在1ml PBS中简单洗涤2次,通过用移液管除去培养基并用新鲜PBS代替。请注意,盖玻片不得干燥。
  3. 使用200μl4%PFA在4℃下10分钟内进行T细胞固定,随后在含有盖玻片的孔中洗涤1ml PBS 1x 2次。
  4. 使用200μl0.1%Triton X-100在室温下2分钟,然后用1ml 1×PBS洗涤2次,获得透化。
  5. 在室温下20分钟内使用1ml 0.5%BSA的PBS封闭盖玻片
  6. 将盖玻片在30至40μl的稀释的一抗(Abs)中在4℃温育过夜(图2)。

    图2. PLA程序的盖玻片处理。A.将直径60mm的塑料培养皿用作Abs温育和PLA实验的湿度室。用填充有水的15ml盖获得湿度。在整个过程中使用平头钳来处理盖玻片。 B.从培养皿中取出后,将盖玻片(*)面向下孵育在30μlAbs(†)上。 C.放置在液滴上的盖玻片(*)。从该步骤,关闭陪替氏培养皿并用石蜡膜密封,并在4℃放置过夜。 D.第二天,使用镊子和针从湿度室小心地拾取盖玻片。然后将盖玻片单独放置在面上在装有3ml 1×PBS的6孔板的孔中洗涤步骤 注意:对于Duolink产品的温育也重复该程序。与制造商的说明相比,它能够节省Duolink ®试剂。仔细跟踪所有其他制造商的说明(孵育时间,洗涤步骤和缓冲液...)
  7. 第2天:在室温下,在1x PBS中将盖玻片洗涤3次,每次5分钟
  8. PLA实验使用制造商的说明书进行。该程序也可在生物协议网站(Lin等人,2015)上直到步骤9.
  9. 使所有样品在室温下在黑暗中干燥
  10. 将一滴含有DAPI的封固剂置于每个样品和载玻片之间。将载玻片保持在4℃下短期储存
  11. 第3天:使用配有适当过滤器(DAPI:Ex 365,Em:BP 445/50; Duolink spot:Ex:BP 550/25,Em:605/70;图3)的63x物镜的荧光显微镜进行成像程序, 。里面的景点核(作为DAPI作为参考)使用Image J及其分析粒子选项卡进行定量。 然后将载玻片保存在-20℃下长期储存。


图3.TH2细胞中Duolink斑点的重现图像。用我们的方案(Nv:Naïve,Scale bar in yellow:5μm)获得的T细胞图像。 Th2细胞中的红点是Duolink斑点。


  1. 尽可能使用新鲜制备的BSA在PBS中,或过滤准备,如果使用旧的解决方案。 如果不考虑这一步骤,沉积物可能出现并干扰斑点检测。


这项工作得到法国国家研究局管理的法国政府拨款的支持,方案为"Investissements d'Avenir",参考文献ANR-11-LABX-0021(Lipstic Labex)。 FG团队是"EquipelabéliséeLigue Nationale Contrele癌症»。 V. D.是"poste d'accueil INSERM"的收件人;该协议改编自Fredriksson等人(2007)和Lin等人(2015)的以前的工作。


  1. Bruchard,M.,Rebe,C.,Derangere,V.,Togbe,D.,Ryffel,B.,Boidot,R.,Humblin,E.,Hamman,A.,Chalmin,F.,Berger, Chevriaux,A.,Limagne,E.,Apetoh,L.,Vegran,F.and Ghiringhelli,F.(2015)。 受体NLRP3是TH2分化的转录调节子。 Nat Immunol 16(8):859-870。
  2. Fredriksson,S.,Dixon,W.,Ji,H.,Koong,A.C.,Mindrinos,M.and Davis,R.W。(2007)。 通过邻近连接进行多重蛋白检测,用于癌症生物标志物验证。/em> 4(4):327-329。
  3. Lin,M.Z.,Martin,J.L。和Baxter,R.C。(2015)。 Proximity ligation assay(PLA)to detect protein-protein interactions in breast cancer。 协议 5(10):e1479。
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引用:Derangère, V., Bruchard, M., Végran, F. and Ghiringhelli, F. (2016). Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells. Bio-protocol 6(10): e1811. DOI: 10.21769/BioProtoc.1811.