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Jun 2021
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Click-iT® Plus OPP Alexa Fluor® Protein Synthesis Assay in Embryonic Cells
在胚胎细胞中Click- iT®Plus OPP Alexa Fluor®蛋白合成分析    

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Abstract

This protocol describes a method to assess relative changes in the level of global protein synthesis in the preimplantation embryo using the Click-iT® Plus OPP Protein Synthesis Assays. In this assay, O-propargyl-puromycin (OPP), an analog of puromycin, is efficiently incorporated into the nascent polypeptide of newly translated proteins in embryonic cells. OPP is fluorescently labeled with a photostable Alexa FluorTM dye and detected with fluorescence microscopy. The intensity of the fluorescence is quantitatively analyzed. This is a fast, sensitive, and non-radioactive method for the detection of protein synthesis in early embryo development. It provides a tool for analyzing the temporal regulation of protein synthesis, as well as the effects of changes in the embryonic microenvironment, and pharmacological and genetic modulations of embryo development.


Graphical abstract:



Figure 1. Brief overview of the procedures of the Click-iT® Plus OPP Alexa Fluor® protein synthesis assay in embryonic cells.

(A) Set up OPP treatments: (1) Set up microdrops containing 50 µL of OPP working solution and label different treatments on the back of culture dishes (e.g., T0, T1, T2, and T3); (2) The drops are overlain with 2–3 mm heavy paraffin oil and then equilibrated in incubator for 2 h; (3) Collect the embryos from female reproductive tracts or following in vitro culture in desired treatments; (4) Culture embryos in the equilibrated OPP working solution for 2–6 h. (B) Example of OPP detection procedures working with 60-well plates labeled as T0, T1, T2, T3, T4, and T5 for different treatments: (1) The first 60-well plate is used for the procedures of washing, fixation, permeabilization, and Click-iT® OPP detection. (2) The second 60-well plate is for DNA staining and washing. (C) Slide preparation: (1) Label the required number of slides and set up vaseline coverslip supports; (2) Add mounting medium; (3) Transfer embryos into mounting medium; (4) Set coverslip; (5) Seal the coverslip with nail polish.

Keywords: Embryo development (胚胎发育), Maternal-embryonic transition (母胎过渡), O-propargyl-puromycin (邻炔丙基嘌呤霉素), Protein synthesis (蛋白质合成)

Background

Fertilization triggers embryonic genome activation, whereby most maternal transcripts and proteins are degraded, followed by the generation of a new transcriptome and resulting proteome (Gao et al., 2017; Svoboda, 2018). These processes are required for the transition from maternal to embryonic control of development and create a cellular environment conducive of the totipotent state of the early embryo. There has been extensive analysis and development of tools for monitoring the changes to transcription at this time, but changes in translation have received less attention. New protein synthesis during the process of maternal to embryonic transition is tightly regulated and can be quantitatively evaluated with several methods, such as 35S-Methionine (Crosby et al., 1988), two-dimensional gel electrophoresis (Latham et al., 1991), and mass spectrometry (Gao et al., 2017). Here we report the development and use of a rapid alternative method for the analysis of global changes in the level of protein synthesis in the early embryo. The Click-iT® Plus OPP Protein Synthesis Assay is a non-toxic and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy and high-throughput imaging. O-propargyl-puromycin (OPP) is a membrane-permeable alkyne puromycin analog that forms covalent bonds with nascent polypeptide chains with cells. The addition of the Alexa Fluor® picolyl azide and the Click reaction reagents leads to a chemoselective ligation or “click” reaction between the picolyl azide dye and the alkyne OPP, and the modified proteins are detected by imaged-based analysis. The assay has been demonstrated to preserve cell morphology and has been used in NIH3T3, HeLa, C2C12 cells, BPAE, U-2 OS, CHO-M1, and A549 (Mateu-Regue et al., 2019; Enam et al., 2020; Liu et al., 2012), and the embryonic cells of preimplantation development (Li et al., 2021).


This protocol is designed to semi-quantitatively assess the relative changes in protein synthesis during the period of mouse maternal to embryonic transition. The first stage is to test and optimize the concentration of the OPP, then confirm the specificity of the assay by use of the known protein synthesis inhibitor Cycloheximide and inhibitor of translation elongation, 4EGI-1 (eIF4E inhibitor) (Li et al., 2021).

Materials and Reagents

  1. Plastic and glass ware

    1. 60-well culture plates (Nunc, Naperville, IL, USA, LUX 5260)

    2. 35 cm × 10 mm Petri dishes (Thermo Scientific, catalog number: 150318)

    3. Cover glasses, 18 × 18 mm (Merck, BR470045)

    4. Microscope slides, 25 mm × 75 mm (Merck, S8902)


  2. Animals

    Six-eight-week-old female mice


  3. Click-iT® Plus OPP Alexa Fluor® protein synthesis assay (Thermo Scientific, catalog number: C10456), including

    1. Component A: Click-iT® OPP Reagent, 20 mM in DMSO

    2. Component B: Alexa Fluor® 488 picolyl azide in DMSO solution

    3. Component C: Click-iT® OPP Reaction Buffer (10× solution containing Tris-buffered saline)

    4. Component D: Copper Protectant

    5. Component E: Click-iT® Reaction Buffer Additive

    6. Component F: Click-iT® Reaction Rise Buffer, containing 2 mM sodium azide.

    7. Component G: NuclearMaskTM Blue Stain, 2,000× concentrate in water


  4. Chemicals and reagents

    1. Dulbecco′s Phosphate Buffered Saline (PBS) (Sigma, catalog number: D5773)

    2. Triton X-100 (Sigma, catalog number: T8787)

    3. Bovine serum albumin (BSA) (Sigma, catalog number: B2064)

    4. Paraformaldehyde (PFA) (Sigma, catalog number: P6148)

    5. DMSO (Sigma, catalog number: D8418)

    6. Click-iT® Plus OPP Alexa Fluor® protein synthesis assay kit (see Recipes)

    7. Fixation buffer (10 mL) (see Recipes)

    8. 1× PBS (50 mL) (see Recipes)

    9. Permeabilization buffer (1 mL) (see Recipes)

    10. Washing solution (10 mL) (see Recipes)

    11. KSOM medium (see Recipes)

Equipment

  1. Dissection microscope (Olympus, SZx7)

  2. Nikon ECLIPSE 80i microscope, Plan Apo 40×/1.0 oil objective (Nikon, Tokyo, Japan)

  3. pH meter SevenDirect SD20 (Mettler Toledo)

  4. Incubator, Cytomat 2 C470-LiN (Themo Fisher Scientific)

Software

  1. ImageJ (http://imagej.net/Fiji)

  2. SPSS for Windows (Version 22.0, SPSS Inc., Chicago, IL, USA)

Procedure

ANIMALS AND TREATMENTS

  1. Animals and embryo collection

    Six-eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU equine chorionic gonadotropin (eCG) (Ningbo Second Hormone Factory, China) and 44–48 h later, 5 IU human chorionic gonadotrophin (hCG) (Livzon, Zhuhai, China), and then placed with fertile males overnight. Pregnancy was confirmed by the presence of a copulation plug in mated females the following morning. Zygotes or two-cell embryos were recovered 20 h and 40 h post-hCG from mated females, respectively. The embryos were collected in HEPES-buffered modified human tubal fluid medium (HEPES-HTF) (O’Neill, 1997), and all components of the media were tissue culture grade (Sigma Chemical Company, St Louis, MO, USA) and contained 3 mg bovine serum albumin/mL (Sigma).


  2. Medium preparation and treatments

    1. Calibrate the concentration of OPP for embryo treatment

      To minimize the embryonic toxicity of OPP during the period of embryo treatment, several doses of OPP should be tested. For example, dilute Component A (20 mM) with KSOM medium (Lawitts and Biggers, 1993) to produce a range of OPP concentrations (e.g., 50, 37.5, and 25 μM) to 400 µL of final working solutions (Table 1).


      Table 1. Prepare a series of working solutions of Click-iT® OPP (Component A)

      OPP (μM) KSOM (µL) Component A (µL) DMSO (µL) Total volume (µL)
      0 399 0 1 400
      25 399 0.5 0.5 400
      37.5 399 0.75 0.25 400
      50 399 1 0 400

      The example is to prepare three doses of OPP. The control is 0 μM OPP in KSOM medium. Adjust the concentration of DMSO to 0.25% (v/v) in all working solutions.

      Note: Total volume can be proportionally adjusted depending on the number of treatments; The concentration of OPP can be readjusted or optimized for the different experimental designs. A range of culture medium types are suitable for use depending upon the experimental design (e.g., hTF medium and KSOM, supplemented with or without amino acids).


    2. OPP treatment (Figures 1 A1-4)

      1. Set up microdrops on culture dishes. Each drop contains 50 µL of OPP working solutions overlaid with 2 mm of heavy paraffin oil (Sigma).

        Optional: use 60-well Terasaki plates (LUX 5260, Nunc, Naperville, IL, USA), each containing 10 µL of OPP working solutions.

      2. Equilibrate in 5% CO2 incubator at 37°C for 2 h.

      3. Transfer the embryos into OPP working solutions.

        Optional: embryo density is optional, e.g., 1–10 embryos in 10 µL of working solutions. Keep the same density in all treatments.

      4. Culture and treat the embryos for 2–6 h at 37°C, with 5% CO2 in air tension.

        CRITICAL: (1) It was suggested that the incubation periods with OPP be between 30 min and 1 h to maximize detection and minimize toxicity (Liu et al., 2012; Signer et al., 2014). However, translation in preimplantation embryos may be different from the somatic cells. In simple formulated medium, 2-cell embryo expressed a peak signal after 6 h treatment with 37.5 µM OPP (Li et al., 2021). Thus, we recommend testing different incubation periods (Figure 2B) with different concentrations of OPP (Figure 2 A) and doses of drugs (Figure 2C) for each stage of embryo development used before the final experimental design is determined (as in the example in Table 1). (2) Similar calibrations are suggested if the experiments are performed in different oxygen concentrations, as this protocol was designed for the OPP treatment in air tension before final experimental design is determined.


STEP-BY-STEP METHOD DETAILS

Note: To facilitate systematically processing embryos, we use 60-well Terasaki plates (Figure 1 B1–2). Each well can contain 10 µL of solution and 1–10 embryos. Transfer embryos with minimal solution into the well of the next step and use clean pipettes for each transfer stage.


  1. Proceed to embryo fixation and permeabilization (Figure 1 B1–2)

    1. Work at room temperature. Transfer embryos treated with OPP into washing solution and rinse once to remove the media.

    2. Transfer embryos into 10 µL fixation buffer/well. Incubate for 15 min at room temperature.

    3. Wash embryos twice with washing solution to remove fixative.

    4. Transfer embryos into 10 µL of permeabilization buffer and incubate for 20 min at room temperature.


  2. Click-iT® OPP Detection (Figure 1 B1–2)

    1. Prepare Click-iT® reaction cocktail according to Table 2.


      Table 2. Click-iT® reaction cocktail

      Reaction components (as supplied in kit) Number of wells
      10 wells 50 wells 100 wells
      Click-iT® OPP Reaction Buffer (1× concentrate) 88 µL 0.44 mL 0.88 mL
      Copper Protectant (Component D) 2 µL 10 µL 20 µL
      Alexa Fluor® picolyl azide (Component B) 0.25 µL 1.25 µL 2.5 µL
      Click-iT® Reaction Buffer Additive (10× solution) 10 µL 50 µL 0.1 mL
      Total reaction volume 0.1 mL 0.5 mL 1 mL

      Note: Use the Click-iT® reaction cocktail within 15 min of preparation. The number of wells can vary depending on the experiment. It is important to calculate the number of wells you will plate before you start the experiment.

    2. Wash embryos twice with 10 µL of washing solution

    3. Transfer embryos into 10 µL of Click-iT® reaction cocktail.

    4. Incubate for 30 min at room temperature. Protected from light.

    5. Wash once with 10 µL per well of Click-iT® reaction rise buffer (Component F).


  3. DNA Staining (Figure 1 B1-2)

    Note: The following protocol is based upon 10 µL of HCS NuclearMaskTM Blue Stain working solution per well.

    1. Dilute HCS NuclearMaskTM Blue Stain (Component G) solution (1:2,000, v/v) in 1× PBS to obtain a 1× HCS NuclearMaskTM Blue Stain working solution.

    2. Transfer embryos into 10 µL of 1× HCS NuclearMaskTM Blue Stain working solution. Incubate for 30 min at room temperature, protected from light.

    3. Wash twice with washing solution and proceed to Imaging and Analysis (below).


  4. Imaging

    1. Make slides (Figure 1 C1–5)

      1. Clean the glass slides and the coverslips with ethanol.

      2. Use an insulin syringe with 18–22 G needle filled with vaseline. Make two parallel lanes of vaseline on the slide to support the coverslip. The vaseline lane should 1–2 mm thick.

      3. Add a drop of 7 µL 1× PBS onto a slide. (Optional: use standard anti-fade mounting media).

      4. Transfer 10 embryos/drop and gently put an 18 × 18 mm coverslip on with forceps; remove any excess fluid from edges of the coverslip with a lint-free absorbent tissue.

      5. Seal with nail polish (optional: PathTech, https://www.pathtech.com.au). Take care not to move the coverslip to avoid squashing the embryos).

    2. Scan the slide under fluorescence microscope with filters appropriate for DAPI/Hoechst and FITC for Alexa Fluor® 488 (Figures 2 A–C).

    3. Whole section image is captured with mercury lamp UV illumination on a Nikon ECLIPSE 80i microscope with a Nikon Plan Apo 40×/1.0 oil objective. (Any similar fluorescent microscope systems are suitable)


  5. Fluorescence measurement and Data analysis

    1. Measure the intensity of the fluorescent OPP stain

      The fluorescent intensity of nascent protein synthesis in the fluorescent channel in the nucleus or whole embryo can be measured by suitable analysis software, e.g., ImageJ and ImagePro Plus (Media Cybernetics, Inc.).

      1. With ImageJ, open the images of the embryo with dual staining of OPP and NuclearMask blue stain.

      2. In the Analyze menu, open Set Measurements and tick “Area”, “Integrated density”, “Mean gray value”, and Standard deviation”.

      3. In the image of NuclearMask blue stain, outline the single nucleus using freehand selection of region of interest (ROI). From Analyze > Tool, open ROI Manager. Add this ROI to ROI Manager and rename it.

      4. Allocate the ROI for the outlined nuclei from ROI Manager to the image of OPP stain of same embryo. This is the exact localization of the nucleus for the dual staining images of same embryo.

      5. Select “Measure” from the analyze menu. The results are shown as a popup box with a stack of values for that nucleus of the embryo cell. The same way to measure the fluorescence of OPP stain of another nucleus.

      6. Directly measure the fluorescent stain of the outlined whole embryo using freehand selection of ROI.

      7. Record and copy the integrated density (stand for the fluorescent intensity of OPP stain) from the popup box for ROI of each nucleus or whole embryo. Set up an Excel spreadsheet for data analysis from all treatments and controls.

      8. Subtract the values in the control treatment of 0 µM OPP from the values from all treatments and controls in the same experiments. The resulting values are used for analysis.

    2. Statistical analysis

      Statistical analysis was performed using SPSS for Windows (SPSS Inc., Chicago, IL, USA) (Optional: other Statistic software). Fluorescence intensity (AU, arbitrary units of optical density of staining) was quantitatively analyzed by univariate analysis of variance. This parameter was set as the dependent variable, while the test treatments and drug doses were the independent variables. Experimental replicates were incorporated into the model as covariates. Differences between individual independent variables were analyzed by the least significance difference test. Less than a 5% probability (P < 0.05) was considered significant.

      Critical: The results from quantitative analysis of fluorescent intensity can vary between experiments due to a range of uncontrollable variables. It is therefore important that embryos from each treatment were processed at the same time and in parallel. All treatments were exposed to the same preparations and dilutions of all reagents. Similarly, all preparations from an experiment were examined microscopically within the same session, and identical microscope and camera settings were used. All image analysis was carried out in an identical manner for each embryo within an experiment. All preparations were carried out by the same experienced operator throughout the study.



    Figure 2. Analysis of OPP staining in the embryonic cells.

    (A) Whole-section images representative of fluorescence changes in wild-type two-cell embryos treated either without or with a range of OPP concentrations for 24 h. There were at least ten embryos in each treatment group. All embryos were arrested at 2-cell stage in the 50 µM OPP group. (B) Two-cell embryos were collected from female reproductive tracts and treated with 37.5 µM OPP for 0, 2, 4, and 6 h. The images are representative of a total of 30 embryos from three independent replicates. (C) Two-cell embryos were treated with either the 4EGI-1 (EIF4E inhibitor) (Li et al., 2021) cycloheximide (CHX) (Schneider-Poetsch et al., 2010) for 6 h. Images are representative of three independent replicates for ten embryos for each treatment. (D) OPP staining intensity in embryos treated with 4EGI-1 or CHX) in (C), compared with the control (no inhibitor). Data are Mean ± S.E.M. (univariate analysis of variance). * Statistically different (P < 0.001) from all other treatments. Scale bars: 10 μm.

Recipes

  1. Click-iT® Plus OPP Alexa Fluor® protein synthesis assay kit

    1. Allow reagent vials to completely thaw and warm to room temperature before opening.

    2. Prior to use, briefly centrifuge Click-iT® OPP Reagent (Component A) and NuclearMaskTM Blue Stain (Component G) to maximize reagent recovery.

    3. Prepare a 10× stock solution of the Click-iT® Reaction Buffer Additive (Component E). Add 2 mL of deionized water to the vial (containing 400 mg) and mix until completely dissolved. After use, aliquot 50 µL/each, store any remaining stock solution at ≤ –20°C.

      Note: When stored as directed, this stock solution is stable for up to 1 year.

    4. Prepare 1× Click-iT® OPP Reaction Buffer.

      Transfer all the solution in the Component C bottle (4 mL) to 36 mL of deionized water. Rinse the Component C bottle with some of the diluted Click-iT® OPP Reaction Buffer to ensure the transfer of all the 10× concentrate.

      Note: Use the Click-iT® reaction cocktail within 15 min of preparation. To prepare smaller amounts of 1× Click-iT® OPP Reaction Buffer, dilute 1 volume from the Component C bottle with nine volumes of deionized water. After use, store any remaining 1× solution at 2°C–8°C. When stored as directed, 1× Click-iT® OPP Reaction Buffer is stable for 6 months.

    5. Prepare working solution of NuclearMaskTM Blue Stain (Component G).

      Prepare it just prior to use. Dilute HCS NuclearMaskTM Blue Stain (Component G) solution 1:2,000 in PBS to obtain a 1× HCS NuclearMaskTM Blue Stain working solution.

  2. Fixation buffer [3.6% (w/v) Paraformaldehyde in PBS] (10 mL)

    1. Weigh Paraformaldehyde 0.37 g.

    2. Add to MilliQ H2O 10 mL in a glass container.

    3. Add 1.0 M NaOH 14 µL.

    4. Warm and stir on a 50–60°C hotplate until completely dissolved. It takes about 20 min.

    5. Add PBS 0.096 g and stir until completely dissolved.

    6. Cover the container and leave until it reaches room temperature.

    7. Adjust pH at 7.4 with 1 N HCl at room temperature.

  3. 1× PBS (50 mL)

    1. Weigh 0.4798 g PBS powder.

    2. Add into 50 mL MilliQ H2O and mix well.

  4. Permeabilization buffer (1 mL)

    1. Prepare prior to use.

    2. 5 µL of Triton X-100

    3. Add into 995 µL of 1× PBS and mix well.

  5. Washing solution (10 mL)

    1. Weigh 0.3 g BSA

    2. Add into 10 mL of 1× PBS. Make sure it is completely dissolved before use.

    Note: Always use the fume hood and follow safety measures when preparing and handling paraformaldehyde. Personal protection equipment should be used during use.

  6. Composition of KSOM medium (100 mL)

    555 mg of NaCl

    18.5 mg of KCl

    4.75 mg of KH2PO4

    4.95 mg of MgSO4•7H2O

    25 mg of CaCl2•2H2O

    210 mg of NaHCO3

    3.6 mg of Glucose

    2.2 mL of Na-Pyruvate

    0.174 mL of DL-Lactic Acid

    4 mg of EDTA

    14.6 mg of GL-Glutamine and 100 mg Bovine serum albumin

    Note: All reagents were purchased from Sigma.

    Add MilliQ purified water to 100 mL, mix well, and sterilize by filtration with 22 mm Millex-GV Filter, 0.22 µm (MERCK).

Acknowledgments

We thank Nanjing Your Bio-tech Development Ltd. Co (Jiangbei New District, Nanjing, Jiangsu Province, China) for the generous donation of all embryonic culture media: KSOM, HEPES-HTF, and HTF.

FUNDING: This work was supported by grants from the National Natural Science Foundation of China awarded to X.J (81471458), and Zhejiang Provincial Natural Science Foundation of China (LQ21H040010) to Y.L.

Competing interests

The authors declare no competing interests

AUTHOR CONTRIBUTIONS: X. Jin. and C.O. supervised the study. X. Jin. C.O. and X.H. designed and wrote the manuscript. Y.L., X.J. L.C., J.T., M.H., and J.L. performed the experiments.

Ethics

Animal experiments were approved by and conducted according to ethics guidelines from relevant research institutes and universities. Hybrid (C57BL/6 X CBA/He) mice were housed and bred at the Wenzhou Medical University.

References

  1. Crosby, I. M., Gandolfi, F. and Moor, R. M. (1988). Control of protein synthesis during early cleavage of sheep embryos. J Reprod Fertil 82(2): 769-775.
  2. Enam, S. U., Zinshteyn, B., Goldman, D. H., Cassani, M., Livingston, N. M., Seydoux, G. and Green, R. (2020). Puromycin reactivity does not accurately localize translation at the subcellular level. Elife 9: e60303.
  3. Gao, Y., Liu, X., Tang, B., Li, C., Kou, Z., Li, L., Liu, W., Wu, Y., Kou, X., Li, J., et al. (2017). Protein Expression Landscape of Mouse Embryos during Pre-implantation Development.Cell Rep 21(13): 3957-3969.
  4. Latham, K. E., Garrels, J. I., Chang, C. and Solter, D. (1991). Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages. Development 112(4): 921-932.
  5. Lawitts, J. A. and Biggers, J. D. (1993). Culture of preimplantation embryos. Methods Enzymol 225: 153-164.
  6. Li, Y., Tang, J., Ji, X., Hua, M. M., Liu, M., Chang, L., Gu, Y., Shi, C., Ni, W., Liu, J., et al. (2021). Regulation of the mammalian maternal-to-embryonic transition by eukaryotic translation initiation factor 4E. Development 148(12).
  7. Liu, J., Xu, Y., Stoleru, D. and Salic, A. (2012). Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin.Proc Natl Acad Sci U S A 109(2): 413-418.
  8. Mateu-Regue, A., Christiansen, J., Bagger, F. O., Winther, O., Hellriegel, C. and Nielsen, F. C. (2019). Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons. Cell Rep 29(3): 736-748 e734.
  9. O'Neill, C. (1997). Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro.Biol Reprod 56(1): 229-237.
  10. Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., Green, R., Shen, B. and Liu, J. O. (2010). Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6(3): 209-217.
  11. Signer, R. A., Magee, J. A., Salic, A. and Morrison, S. J. (2014). Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509(7498): 49-54.
  12. Svoboda, P. (2018). Mammalian zygotic genome activation.Semin Cell Dev Biol 84: 118-126.

简介

[摘要]本协议描述了一种使用 Click- iT ® Plus OPP 蛋白质合成测定法评估植入前胚胎中整体蛋白质合成水平相对变化的方法。在该测定中,嘌呤霉素的类似物O-炔丙基嘌呤霉素(OPP) 有效地掺入胚胎细胞中新翻译蛋白质的新生多肽中。 OPP用光稳定的 Alexa Fluor TM染料进行荧光标记,并用荧光显微镜检测。定量分析荧光强度。这是一种快速、灵敏且无放射性的方法,用于检测早期胚胎发育中的蛋白质合成。它提供了一种工具来分析蛋白质合成的时间调节,以及胚胎微环境变化的影响,以及胚胎发育的药理学和遗传调节。

图形概要:


Click- iT ® Plus OPP Alexa Fluor ®在胚胎细胞中的蛋白质合成测定程序的简要概述。
( A ) 设置 OPP 处理: (1)设置含有 50 µL OPP 工作溶液的微滴,并在培养皿背面标记不同的处理(例如,T0、T1、T2 和 T3); (2 )液滴上覆盖2-3 mm 重质石蜡油,然后在培养箱中平衡 2 h; (3)从雌性生殖道采集胚胎或进行所需处理后的体外培养; (4)在平衡过的 OPP 工作液中培养胚胎2-6小时。 ( B ) OPP 检测程序示例使用标记为 T0、T1、T2、T3、T4 和 T5 的 60 孔板进行不同处理: (1) 第一个 60 孔板用于洗涤、固定程序、透化和Click- iT ® OPP 检测。 (2)第二个 60 孔板用于 DNA 染色和洗涤。 (C)载玻片准备: (1) 标注所需的载玻片数量并设置凡士林盖玻片支架; (2) 加入封固剂; (3) 将胚胎转移到封固剂中; (4) 设置盖玻片; (5) 用指甲油密封盖玻片。


[背景]受精触发胚胎基因组激活,大多数母体转录物和蛋白质被降解,随后产生新的转录组和蛋白质组(Gao et al. , 2017; Svoboda, 2018) 。这些过程是从母体到胚胎控制发育的转变所必需的,并创造了一个有利于早期胚胎全能状态的细胞环境。目前已经对用于监控转录变化的工具进行了广泛的分析和开发,但翻译的变化受到的关注较少。母体向胚胎转变过程中的新蛋白质合成受到严格调控,可以通过多种方法进行定量评估,例如35 S-蛋氨酸(Crosby et al. , 1988) 、二维凝胶电泳(Latham et al. , 1991) )和质谱法(Gao et al. , 2017) 。在这里,我们报告了一种快速替代方法的开发和使用,用于分析早期胚胎中蛋白质合成水平的整体变化。 Click - iT ® Plus OPP 蛋白质合成分析是一种无毒和非放射性的方法,用于利用荧光显微镜和高通量成像检测新生蛋白质合成。 O-炔丙基嘌呤霉素 (OPP) 是一种膜可渗透的炔嘌呤霉素类似物,可与细胞中的新生多肽链形成共价键。添加Alexa Fluor® 吡啶甲基 叠氮化物和点击反应试剂导致吡啶甲基之间的化学选择性连接或“点击”反应 叠氮化物染料和炔烃 OPP,以及修饰的蛋白质通过基于成像的分析进行检测。该测定已被证明可以保持细胞形态,并已用于 NIH3T3、HeLa、C2C12 细胞、BPAE、U-2 OS、CHO-M1 和 A549 (Mateu-Regue等人,2019 年; Enam等人,2020 年) ; Liu et al. , 2012) ,以及胚胎着床前发育的细胞(Li et al. , 2021) 。
该协议旨在半定量评估小鼠母体向胚胎过渡期间蛋白质合成的相对变化。第一阶段是测试和优化 OPP 的浓度,然后通过使用已知的蛋白质合成抑制剂放线菌酮和翻译延伸抑制剂 4EGI-1(eIF4E 抑制剂)确认测定的特异性(Li et al. , 2021 ) .

关键字:胚胎发育, 母胎过渡, 邻炔丙基嘌呤霉素, 蛋白质合成



材料和试剂


A.塑料和玻璃器皿
1.60 孔培养板 ( Nunc, Naperville, IL, USA, LUX 5260)
2.35 cm × 10 mm 培养皿( Thermo Scientific,目录号: 150318)
3.盖玻片,18 × 18 毫米(默克,BR470045)
4.显微镜载玻片,25 mm × 75 mm(Merck,S8902)


B.一种动物
六八周大的雌性老鼠


C.Click - iT® Plus OPP Alexa Fluor®蛋白质合成测定( Thermo Scientific,目录号:C10456 ) ,包括
1.组分 A:Click- iT ® OPP 试剂,20 mM DMSO
2.组分 B:Alexa Fluor ® 488吡啶甲基 DMSO溶液中的叠氮化物
3.组分 C:Click - iT ® OPP 反应缓冲液(10 ×含有 Tris 缓冲盐水的溶液)
4.组分 D:铜保护剂
5.组分 E:Click- iT® Reaction Buffer Additive
6.组分 F:Click- iT® Reaction Rise Buffer,含有 2 mM叠氮化钠。
7.组分 G: NuclearMask TM Blue Stain,2,000 ×浓缩水


D.化学品和试剂
1.Dulbecco的磷酸盐缓冲盐水(PBS) (Sigma,目录号: D5773)
2.Triton X-100 (Sigma,目录号: T8787)
3.牛血清白蛋白(BSA) (Sigma,目录号: B2064)
4.多聚甲醛(PFA) (Sigma,目录号: P6148)
5.DMSO(Sigma,目录号:D8418)
6.Click- iT ® Plus OPP Alexa Fluor ®蛋白质合成检测试剂盒(见配方)
7.固定缓冲液(10 mL)(见配方)
8.1 × PBS (50 mL)(见配方)
9.透化缓冲液(1 mL)(见配方)
10.洗涤液(10 mL)(见配方)
11.KSOM 培养基(见食谱)


设备


1.解剖显微镜(Olympus,SZx7)
2.尼康 ECLIPSE 80i 显微镜,Plan Apo 40 × /1.0 油镜(尼康,东京,日本)
3.pH计SevenDirect SD20 (梅特勒-托利多)
4.孵化器, Cytomat 2 C470-LiN(Themo Fisher Scientific)




软件


1.ImageJ ( http://imagej.net/Fiji )
2.SPSS for Windows(22.0 版, SPSS Inc.,Chicago,IL,USA)


程序


动物和治疗


A.动物和胚胎采集
六八周龄雌性小鼠通过腹腔注射 5 IU 马绒毛膜促性腺激素 ( eCG ) (中国宁波第二激素厂)和 44-48小时后,5 IU 人绒毛膜促性腺激素 ( hCG )(珠海丽珠)进行超排卵,中国),然后与有生育能力的雄性一起放置过夜。第二天早上,已交配的雌性中存在交配插头,从而证实了怀孕。受精卵或双细胞胚胎分别在hCG后 20 小时和 40 小时从已交配的雌性中回收。将胚胎收集在 HEPES 缓冲的改良人输卵管液培养基 (HEPES-HTF) (O'Neill, 1997)中,培养基的所有成分均为组织培养级 (Sigma 美国密苏里州圣路易斯化学公司)并含有 3 mg 牛血清白蛋白/mL (Sigma)。


B.培养基制备和处理
1.校准用于胚胎处理的 OPP 浓度
为了在胚胎治疗期间尽量减少 OPP 的胚胎毒性,应测试几剂 OPP。例如,用 KSOM 培养基(Lawitts 和 Biggers,1993 年)稀释组分 A(20 mM)以产生一系列 OPP 浓度(例如,50、37.5 和 25 μM )至 400 μL 的最终工作溶液(表 1)。


表 1.准备 Click- iT ® OPP(组分 A)的一系列工作解决方案
示例是准备三剂 OPP。对照为 KSOM 培养基中的 0 μM OPP。在所有工作溶液中将 DMSO 的浓度调整为 0.25% (v/v)。
注:总量可根据治疗次数按比例调整; OPP 的浓度可以针对不同的实验设计进行重新调整或优化。根据实验设计,可以使用多种培养基类型(例如, hTF培养基和 KSOM,添加或不添加氨基酸) 。
2.OPP 处理(图 1 A1-4)
a.在培养皿上设置微滴。每滴含有 50 µL 的 OPP 工作溶液,上面覆盖有 2 mm 的重质石蜡油 (Sigma)。
可选:使用 60 孔Terasaki板(LUX 5260,Nunc,Naperville,IL,USA),每个板含有 10 μL OPP 工作溶液。
b.在 37 °C的5% CO 2培养箱中平衡2 小时。
c.将胚胎转移到 OPP 工作解决方案中。
可选:胚胎密度是可选的,例如,10 µL 工作溶液中有 1 – 10 个胚胎。在所有处理中保持相同的密度。
d.在 37 °C 下培养和处理胚胎2-6 小时,空气张力为5% CO 2 。
关键:(1) 建议 OPP 的潜伏期在 30 分钟到 1 小时之间,以最大限度地提高检测率并最大限度地降低毒性(Liu 等人,2012 年;Signer 等人,2014 年) 。然而,植入前胚胎中的翻译可能与体细胞不同。在简单配制的培养基中,2 细胞胚胎在用 37.5 µM OPP 处理 6 小时后表现出峰值信号(Li et al., 2021) 。因此,我们建议在确定最终实验设计之前使用不同浓度的 OPP (图 2A)和药物剂量(图 2C)测试不同的孵化期(图 2B) ,用于胚胎发育的每个阶段(如示例表格1)。 (2) 如果实验是在不同的氧气浓度下进行的,建议进行类似的校准,因为该协议是为在最终实验设计确定之前在空气张力下进行 OPP 处理而设计的。


分步方法详细信息
注意:为了便于系统地处理胚胎,我们使用 60 孔Terasaki板(图 1 B1 – 2)。每口井可包含 10 µL 溶液和 1 – 10 个胚胎。将含有最少溶液的胚胎转移到下一步的孔中,并在每个转移阶段使用干净的移液器。


A.进行胚胎固定和透化(图 1 B1 – 2)
1.在室温下工作。将用 OPP 处理的胚胎转移到洗涤液中并冲洗一次以去除培养基。
2.将胚胎转移到 10 μL 固定缓冲液/孔中。孵育 15 分钟 在室温下。
3.用洗涤液清洗胚胎两次以去除固定剂。
4.将胚胎转移到 10 μL 的透化缓冲液中并孵育 20 分钟 在室温下。


B.Click- iT ® OPP 检测(图 1 B1 – 2)
1.根据表2制备 Click - iT®反应混合物。


表 2. Click- iT ®反应混合物


注意:在制备后 15 分钟内使用 Click- iT®反应混合物。孔的数量可能因实验而异。在开始实验之前计算您将电镀的孔数很重要。
2.用 10 μL 的洗涤液清洗胚胎两次
3.将胚胎转移到 10 µL Click- iT ®反应混合物中。
4.在室温下孵育 30 分钟。避光。
5.每孔用 10 µL Click - iT ®反应上升缓冲液(组分 F)清洗一次。


C.DNA 染色(图 1 B1-2)
注意:以下协议基于每口井 10 µL HCS NuclearMask TM Blue Stain 工作溶液。
1.× PBS 中稀释 HCS NuclearMask TM Blue Stain(组分 G)溶液(1:2,000,v/v),以获得 1 × HCS NuclearMask TM Blue Stain 工作溶液。
2.将胚胎转移到 10 μL 的 1 × HCS NuclearMask TM蓝色染色工作溶液中。在室温下孵育 30 分钟,避光。
3.用洗涤液洗涤两次,然后进行成像和分析(下)。


D.成像
1.制作幻灯片(图 1 C1 – 5)
a.用乙醇清洁载玻片和盖玻片。
b.使用装有凡士林的 18-22 G 针头的胰岛素注射器。在幻灯片上制作两条平行的凡士林通道以支撑盖玻片。凡士林通道应为1-2 毫米厚。
c.添加一滴 7 μL 1 × PBS。 (可选:使用标准的防褪色安装介质)。
d.转移 10 个胚胎/滴,然后用镊子轻轻盖上 18 × 18 mm 盖玻片;用无绒吸水纸巾去除盖玻片边缘多余的液体。
e.用指甲油密封(可选: PathTech , https ://www.pathtech.com.au )。注意不要移动盖玻片以避免挤压胚胎)。
2.在荧光显微镜下使用适用于 DAPI/Hoechst 的滤光片和适用于 Alexa Fluor ® 488 的 FITC 扫描载玻片(图 2 A - C)。
3.× /1.0 油物镜上使用汞灯紫外照明捕获整个截面图像。 (任何类似的荧光显微镜系统都适用)


E.荧光测量和数据分析
1.测量荧光 OPP 染色的强度
可以通过合适的分析软件,例如ImageJ和ImagePro Plus(Media Cybernetics,Inc.)测量细胞核或整个胚胎中荧光通道中新生蛋白质合成的荧光强度。
a.NucleMask蓝染色双重染色的胚胎图像。
b.在“分析”菜单中,打开“设置测量”并勾选“面积”、“积分密度”、“平均灰度值”和“标准差”。
c.在NuclearMask蓝色染色图像中,勾勒出 使用徒手选择感兴趣区域(ROI) 的单核。从分析 > 工具,打开 ROI 管理器。将此 ROI 添加到 ROI Manager 并重命名。
d.将 ROI 管理器中概述的核的 ROI 分配到同一胚胎的 OPP 染色图像。这是同一胚胎的双重染色图像的细胞核的精确定位。
e.从分析菜单中选择“测量”。结果显示为一个弹出框,其中包含胚胎细胞核的一堆值。用同样的方法测量另一个核的 OPP 染色的荧光。
f.使用徒手选择 ROI 直接测量概述的整个胚胎的荧光染色。
g.从弹出框中记录并复制每个核或整个胚胎的 ROI 的综合密度(代表 OPP 染色的荧光强度)。设置 Excel 电子表格,用于分析所有处理和控制的数据。
h.从同一实验中所有处理和控制的值中减去 0 μM OPP 控制处理中的值。结果值用于分析。
2.统计分析
使用 SPSS for Windows(SPSS Inc.,Chicago,IL,USA)(可选:其他统计软件)进行统计分析。通过单变量方差分析定量分析荧光强度(AU,染色光密度的任意单位)。该参数设置为因变量,而测试治疗和药物剂量为自变量。实验重复作为协变量被纳入模型。通过最小显着性差异检验分析个体自变量之间的差异。小于 5% 的概率 (P < 0.05) 被认为是显着的。
关键:由于一系列不可控的变量,荧光强度定量分析的结果可能因实验而异。因此,重要的是每次处理的胚胎同时并并行处理。所有处理均暴露于所有试剂的相同制剂和稀释液。同样,实验中的所有准备工作都在同一会话中进行了显微镜检查,并使用了相同的显微镜和相机设置。对于实验中的每个胚胎,所有图像分析都以相同的方式进行。在整个研究过程中,所有准备工作均由同一位经验丰富的操作员进行。




图 2. 胚胎细胞中 OPP 染色分析。
(A)代表野生型双细胞胚胎荧光变化的全截面图像,无论是在没有或使用一系列 OPP 浓度的情况下处理 24 小时。每个治疗组至少有十个胚胎。在 50 µM OPP 组中,所有胚胎都在 2 细胞阶段被逮捕。 (B)从女性生殖道收集双细胞胚胎,并用 37.5 μM OPP 处理 0、2、4 和 6 小时。这些图像代表了来自三个独立复制的总共 30 个胚胎。 (C) 用 4EGI-1 (EIF4E 抑制剂) (Li et al. , 2021)环己酰亚胺 (CHX) (Schneider-Poetsch et al. , 2010)处理双细胞胚胎6 小时。图像代表每次处理的十个胚胎的三个独立重复。 (四) (C)中用4EGI-1或CHX)处理的胚胎中的OPP染色强度与对照(无抑制剂)相比。数据是平均值±SEM(单变量方差分析)。 * 与所有其他治疗有统计学差异 (P < 0.001)。比例尺:10 μm 。 


食谱


1.Click- iT ® Plus OPP Alexa Fluor ®蛋白质合成检测试剂盒
a.打开前,让试剂瓶完全解冻并升温至室温。
b.Click- iT ® OPP 试剂(组分 A)和NuclearMask TM Blue Stain(组分 G)短暂离心,以最大限度地提高试剂回收率。
c.准备Click- iT ® Reaction Buffer Additive(组分 E)的 10倍储备溶液。将 2 mL 去离子水加入小瓶(含 400 mg)并混合直至完全溶解。使用后,分装 50 µL/每个,将剩余的储备溶液储存在 ≤ –20°C。
注意:按说明储存时,该储备溶液可稳定保存长达 1 年。
d.准备 1 × Click- iT ® OPP 反应缓冲液。
将组分 C 瓶 (4 mL) 中的所有溶液转移到 36 mL 的去离子水中。用一些稀释的 Click- iT ® OPP 反应缓冲液冲洗组分 C 瓶,以确保转移所有 10 ×浓缩液。
注意:在制备后 15 分钟内使用 Click- iT ®反应混合物。要制备更少量的 1 × Click- iT ® OPP 反应缓冲液,请用 9 倍体积的去离子水从 Component C 瓶中稀释 1 倍体积。使用后,将剩余的 1 ×溶液储存在 2°C–8°C。按指示储存时,1 × Click- iT ® OPP Reaction Buffer 可稳定保存 6 个月。
e.准备NuclearMask TM Blue Stain(组分G)的工作溶液。
在使用前准备好。在 PBS 中按 1:2,000稀释 HCS NuclearMask TM Blue Stain(组分 G)溶液,以获得 1 × HCS NuclearMask TM Blue Stain 工作溶液。
2.固定缓冲液 [3.6% (w/v) 多聚甲醛在 PBS] (10 mL)
a.称取0.37 克多聚甲醛。
b.在玻璃容器中加入MilliQ H 2 O 10 mL。
c.添加 1.0 M NaOH 14 µL。
d.在 50 – 60 °C加热板上加热并搅拌直至完全溶解。大约需要20分钟。
e.添加 PBS 0.096 g 并搅拌直至完全溶解。
f.盖上容器并离开,直到它达到室温。
g.在室温下用 1 N HCl 将 pH 值调节到 7.4。
3.1 × PBS(50 毫升)
a.称量 0.4798 克 PBS 粉末。
b.加入 50 mL MilliQ H 2 O 并充分混合。
4.透化缓冲液 (1 mL)
a.使用前准备。
b.5 µL 海卫 X-100
c.添加到 995 μL的 1 × PBS 中并充分混合。
5.洗涤液(10 毫升)
a.称重 0.3 克 BSA
b.添加到 10 mL 的 1 × PBS 中。确保在使用前完全溶解。
注意:在制备和处理多聚甲醛时,始终使用通风柜并遵循安全措施。使用过程中应使用个人防护装备。
6.KSOM 培养基 (100 毫升) 的组成
555 毫克氯化钠
18.5 毫克氯化钾
4.75 毫克 KH 2 PO 4
4.95 mg MgSO 4 •7H 2 O
25 mg CaCl 2 •2H 2 O
210 毫克碳酸氢钠3
3.6毫克葡萄糖
2.2 毫升丙酮酸钠
0.174 mL DL-乳酸
4 毫克 EDTA
14.6 毫克 GL-谷氨酰胺和 100 毫克牛血清白蛋白
注:所有试剂均购自 Sigma。
加入MilliQ纯化水至 100 mL,充分混合,用 22 mm 过滤除菌 Millex -GV 过滤器,0.22 µm (MERCK)。


致谢


我们感谢南京优尔生物科技发展有限公司(中国江苏省南京市江北新区)慷慨捐赠所有胚胎培养基:KSOM、HEPES-HTF 和 HTF。
FUNDING:这项工作得到了中国国家自然科学基金授予 XJ (81471458) 和中国浙江省自然科学基金( LQ21H040010 )授予 YL的资助


利益争夺 


作者声明没有竞争利益
作者贡献: X. Jin 。和 CO 监督了这项研究。十进。 CO 和 XH 设计并撰写了手稿。 YL、XJLC、JT、MH 和 JL 进行了实验。


伦理


动物实验经相关研究机构和大学的伦理准则批准并根据其进行。杂交(C57BL/6 X CBA/He)小鼠在温州医科大学饲养和繁殖。


参考


1.Crosby, IM, Gandolfi, F. 和 Moor, RM (1988)。绵羊胚胎早期卵裂过程中蛋白质合成的控制。 J Reprod Fertil 82(2):769-775。
2.Enam, SU, Zinshteyn, B., Goldman, DH, Cassani, M., Livingston, NM, Seydoux, G. 和 Green, R. (2020)。嘌呤霉素反应性不能准确定位亚细胞水平的翻译。 生命9 : e60303 。
3.高,Y.,刘,X.,唐,B.,李,C.,寇,Z.,李,L.,刘,W.,吴,Y.,寇,X.,李,J.,等人。 (2017)。植入前发育过程中小鼠胚胎的蛋白质表达情况。 细胞代表21(13):3957-3969。
4.Latham, KE, Garrels, JI, Chang, C. 和 Solter, D. (1991)。小鼠胚胎中蛋白质合成的定量分析。 I. 单细胞和双细胞阶段的广泛重编程。 发展112(4):921-932。
5.Lawitts, JA 和 Biggers, JD (1993)。植入前胚胎的培养。 方法 Enzymol 225:153-164。
6.Li, Y., Tang, J., Ji, X., Hua, MM, Liu, M., Chang, L., Gu, Y., Shi, C., Ni, W., Liu, J., et人_ (2021 年)。通过真核翻译起始因子 4E 调节哺乳动物母体到胚胎的转变。 发展148(12)。
7.Liu, J.、Xu, Y.、Stoleru, D. 和 Salic, A. (2012)。用嘌呤霉素的炔烃类似物对细胞和组织中的蛋白质合成进行成像。 Proc Natl Acad Sci USA 109(2):413-418。
8.Mateu-Regue, A.、Christiansen, J.、Bagger, FO、Winther, O.、Hellriegel, C. 和 Nielsen, FC (2019)。单个 mRNP 分析表明,小的细胞质 mRNP 颗粒代表 mRNA 单例。 细胞代表29(3):736-748 e734。
9.奥尼尔,C. (1997)。小鼠体外植入前胚胎发育需要自分泌生长因子的证据。 Biol Reprod 56(1):229-237。
10.Schneider-Poetsch, T., Ju, J., Eyler, DE, Dang, Y., Bhat, S., Merrick, WC, Green, R., Shen, B. 和 Liu, JO (2010)。放线菌酮和乳酰亚胺霉素抑制真核翻译延伸。 自然化学生物学6(3): 209-217。
11.Signer, RA, Magee, JA, Salic, A. 和 Morrison, SJ (2014)。造血干细胞需要高度调节的蛋白质合成速率。 自然509(7498):49-54。
12.斯沃博达,P. (2018)。哺乳动物合子基因组激活。 精细胞开发生物学84:118-126。


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引用:Li, Y., Ji, X., Chang, L., Tang, J., Hua, M. M., Liu, J., O'Neill, C., Huang, X. and Jin, X. (2022). Click-iT® Plus OPP Alexa Fluor® Protein Synthesis Assay in Embryonic Cells. Bio-protocol 12(11): e4441. DOI: 10.21769/BioProtoc.4441.
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