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Notch Ligand Binding Assay Using Flow Cytometry
使用流式细胞术的Notch配体结合检测   

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eLIFE
Apr 2017

Abstract

Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.

Keywords: Notch ligand binding assay (Notch配体结合测定), DLL1 (DLL1), DLL4 (DLL4), JAG1 (JAG1), JAG2 (JAG2), Fc-tag (Fc标签), Flow cytometry (流式细胞术)

Background

Cell proliferation, differentiation, and apoptosis are well known to be regulated by Notch signaling. Aberrant changes in Notch signaling are related to diverse disorders, giving rise to a range of developmental and adult diseases (Bray, 2016). The canonical Notch signaling pathway in mammals is initiated by the binding of Notch ligands Delta or Jagged to the extracellular domain of Notch receptors (NECD), expressed on opposing cells. Receptor-ligand binding initiates two sequential proteolytic cleavages, resulting in the release of the Notch intracellular domain (NICD). Released NICD complexes with the transcriptional repressor CSL (CBF-1/Suppressor-of-hairless/Lag-1), also termed recombination signal binding protein for immunoglobulin kappa J region (RBPjk), and the co-activator Mastermind (MAML), activate Notch target genes. The binding of Notch receptors to different ligands results in distinct consequences (Benedito et al., 2009; Bray, 2016). For example, the maintenance of hematopoietic stem cells is regulated by low strength JAG1-mediated Notch signaling, whereas arterial cell fate is determined by high strength DLL4-mediated Notch signaling (Gama-Norton et al., 2015). The addition of N-acetylglucosamine (GlcNAc) to the O-fucose on epidermal growth factor (EGF) repeats of the NECD by a Fringe glycosyltransferase generally enhances signaling by Notch receptors induced by Delta-like ligands DLL1 and DLL4, while reducing signaling induced by Jagged ligands JAG1 and JAG2 (Bruckner et al., 2000; Moloney et al., 2000; Yang et al., 2005; Kovall et al., 2017). Recent structural studies have revealed molecular interactions between O-glycans on a Notch1 fragment including EGF repeats 8-13, and soluble ligands DLL4 (Luca et al., 2015) and JAG1 (Luca et al., 2017). Notch ligand EGF repeats are also modified with O-glycans but mutant ligands lacking O-glycans largely remain functional (Muller et al., 2014; Serth et al., 2015). The protocol described below is a method of determining the relative binding of soluble Notch ligand ECDs to the ECD of endogenous or introduced Notch receptors (Figure 1).


Figure 1. Diagram of the Notch ligand binding assay. Notch receptors expressed on the cell surface have an extracellular domain (NECD) comprised of 29-36 N-terminal EGF like repeats followed by 3 Lin-12 Notch repeats. Cell surface expression is confirmed using anti-NECD antibodies. NECD is non-covalently attached to the intracellular domain (NICD) which gets released from the cell membrane and translocates to the nucleus upon proteolysis following ligand binding. Notch ligands Delta-like (DLL1 and DLL4) and Jagged (JAG1 and JAG2) extracellular domains (ECD) comprise a Module at the N-terminus of Notch Ligand (MNNL) motif, followed by a Delta-Serrate-LAG2 (DSL) domain, followed by 6-16 EGF repeats. For this assay, the C-terminus of Notch ligand ECD is linked to a human Fc-tag which is recognized by a fluorescently-labeled secondary antibody (PE-Ab). Ligand binding buffer must contain calcium for Notch ligand binding to occur. Chelation of calcium is used as a control for the specificity of ligand binding.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1110-3000 , 1110-1000 , 1111-2021 )
  2. T75 flask (Corning, Falcon®, catalog number: 353136 ) or 100 mm TC-treated Tissue Culture Dish (Corning, Falcon®, catalog number: 353003 )
  3. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352099 )
  4. 1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
  5. Aluminum foil (Fisher Scientific, catalog number: 01-213-104 )
  6. 5 ml polystyrene round-bottom tube with cell strainer cap (Corning, Falcon®, catalog number: 352235 )
  7. Syringe derived filtered units, 0.22 µm (Merck, catalog number: SLGV013SL )
  8. Amicon Centrifugal filter units, 30K (Merck, catalog number: UFC503024 )
  9. Enzyme-free cell dissociation solution (Merck, catalog number: S-014-B )
  10. Alpha MEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11900073 )
  11. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
  12. Fc Receptor (FcR) block–purified rat-anti-mouse CD16/CD32, clone 2.4G2 (mouse BD Fc block) (BD, BD Biosciences, catalog number: 553141 )
  13. Notch ligands: DLL1, DLL4, JAG1 and JAG2 ECD with a human Fc domain tag at the C-terminus produced from HEK293T cells stably expressing ligand-Fc constructs as described previously (Stahl et al., 2008). Alternatively, soluble, Fc-tagged Notch ligands can be purchased from R&D Systems:
    DLL1 (R&D Systems, catalog number: 5026-DL-050 )
    JAG1 (R&D Systems, catalog number: 599-JG-100 )
    JAG2 (R&D Systems, catalog number: 4748-JG-050 )
    DLL4 (Thermo Fisher Scientific, catalog number: 10171H02H25 )
  14. Secondary antibody: R-Phycoerythrin (PE) AffiniPure F(ab’)2 fragment goat-anti-human IgG, Fcγ fragment-specific (Jackson ImmunoResearch, catalog number: 109-116-170 )
  15. Sodium chloride (Fisher Scientific, catalog number: S271 )
  16. Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S3264 )
  17. Potassium phosphate monobasic (Fisher Scientific, catalog number: P285 )
  18. Potassium chloride (Fisher Scientific, catalog number: P217 )
  19. Magnesium chloride hexahydrate (Fisher Scientific, catalog number: M33 )
  20. Calcium chloride dihydrate (Sigma-Aldrich, catalog number: C3881 )
  21. Hanks’ balanced salt solution (HBSS) (Mediatech, catalog number: 55-022-PB )
  22. Bovine serum albumin (BSA) (Gemini Bio-Products, catalog number: 700-100P )
  23. Sodium azide (Fisher Scientific, catalog number: BP922I-500 )
  24. 16% paraformaldehyde (PFA) in aqueous solution (Electron Microscopy Sciences, catalog number: 15710 )
  25. Pro293aTM (Lonza, catalog number: 12-764Q )
  26. PBS with cations, pH 7.2-7.4 (see Recipes)
  27. Ligand binding buffer (LBB), pH 7.2-7.4 (see Recipes)
  28. 4% PFA in PBS (see Recipes)
  29. Soluble Notch ECD ligand with Fc tag (see Recipes)

Equipment

  1. Pipettes (Mettler-Toledo, Rainin, catalog numbers: 17008653 , 17008650 , 17008649 ; Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641070N )
  2. Benchtop centrifuge (GMI, IEC, model: HN-SII )
  3. Benchtop centrifuge (Eppendorf, model: 5417 C )
  4. Coulter Particle Counter (Beckman Coulter, model: Z1 Series )
  5. Flow cytometer (Cytek Biosciences, model: DxP 10 )

Software

  1. Acquisition Software: FlowJo CE 7.5.110.7
  2. Analysis Software: FlowJo version 10.3.0.Beta3

Procedure

  1. For adherent cells
    1. Remove culture medium and wash the cell layer once with 5 ml of cold PBS (with cations; see Recipes) at room temperature (RT).
    2. Remove PBS and add 1 ml of enzyme-free dissociation reagent per T75 flask or 10 cm dish to dissociate the cells at RT.
    3. Transfer the flask or dish to 37 °C. After 1 min (minute), check if cells have started to detach. If not, keep them at 37 °C until detachment is obvious.
    4. Vigorously tap the sides of the flask or dish to dissociate the cells.
    5. After most cells have detached, re-suspend the cells in 9 ml of medium containing 10% FBS to obtain a single cell suspension.
    6. Adherent cells that are difficult to dissociate using enzyme-free dissociation reagent, the cells can be scraped off the flask or dish and resuspended as a single cell suspension in medium containing 10% FBS. Be careful allow clumped cells to settle.
  2. For single cell suspension obtained above and for cells growing in suspension
    1. Count the cells–need at least 2 x 106 cells per ligand per replicate, if using 0.5 x 106 cells per reaction (0.5 x 106 cells: unstained cells to set the flow cytometer, 0.5 x 106 cells for negative control, 106 cells for test samples [Notch ligand-Fc]).
    2. Centrifuge the required cell volume in a 15 ml Falcon tube at 115 x g (1,000 rpm) for 10 min in a benchtop centrifuge (GMI, IEC, model: HN-SII) at RT.
    3. Aspirate the supernatant and wash the cell pellet in 10 ml ligand LBB (see Recipes). Be careful not to aspirate the cell pellet.
  3. Fix cells in 4% PFA
    1. Centrifuge cells as above and aspirate supernatant.
    2. Add 1 ml of 4% PFA per 107 cells to the cell pellet and gently resuspend by vortexing in brief spurts in 4% PFA (see Recipes).
    3. Incubate cells in 4% PFA for 10 min at RT.
    4. Centrifuge at 115 x g (1,000 rpm) for 10 min and discard supernatant.
    5. Wash the cells twice more with 10 ml LBB.
    6. Resuspend cells to a final concentration of 106 cells/ml in LBB.
    7. Fixed cells can be stored at 4 °C for at least a month. The advantage of using fixed cells is that endocytosis of membrane receptors cannot occur, different cell types can be prepared and fixed on different days, and the ligand binding assay can be performed on all samples with each Notch ligand-Fc on the same day in one experiment, thereby reducing variation between samples.
    8. Unfixed cells can also be used for Notch ligand binding experiments. However, the cells should be used fresh from exponentially growing cultures at 37 °C, and washed in cold PBS, prior to assaying binding at 4 °C. It is important that endocytosis of Notch receptors is prevented and sodium azide at 0.05% can be included in the binding assay for that purpose. Cells that have a damaged plasma membrane can be identified using dyes like 7-amino actinomycin D (7-AAD), Hoechst 33342 and 4,6-diamidino-2-phenylindole (DAPI) in LBB added to cells just prior to flow cytometry, and subsequently gated out of the cells to be analyzed.
  4. Experimental design
    Label 1.5 ml Eppendorf tubes and aliquot a fixed number of cells based on the following experimental design.
    1. Controls for the experiment:
      1. Unstained cells for background fluorescence: A mixture of equal numbers of each cell type to be assayed is aliquoted into a 1.5 ml Eppendorf tube so the final cell number is 0.5-1.0 x 106 cells in LBB. Mix briefly by vortexing. This sample is used to establish parameters in the flow cytometer.
      2. Negative controls-Fc tag alone or secondary antibody alone: Take an aliquot with 0.5-1.0 x 106 cells in LBB into a 1.5 ml Eppendorf tube for subsequent incubation with control-Fc or secondary antibody alone. A negative control is set up for each cell type.
    2. Test samples: Different Notch ligands tagged with Fc
      For each Notch ligand-Fc, aliquot 0.5-1.0 x 106 cells in LBB into separate 1.5 ml Eppendorf tubes.
  5. Centrifuge cells aliquoted above at 420 x g (2,000 rpm) in a benchtop centrifuge (Eppendorf, model: 5417 C) for 5 min at 4 °C, and discard the supernatant.
  6. Wash cells with 1 ml ice cold LBB.
  7. Repeat the wash once and discard the supernatant.
  8. Add 50 μl FcR block (diluted 1:50 in LBB) to all cells, except the unstained control cells, which receive 50 μl LBB, and gently vortex.
  9. Incubate for 15 min on ice. Do not wash.
  10. Add 100 μl Notch ligand-Fc or Fc-tag alone (100-1,000 ng in 100 μl LBB). To unstained cells, or cells that will receive secondary antibody alone, add 100 μl LBB. Gently vortex each tube to mix.
  11. Incubate for 1 h on ice with intermittent mixing by hand every 15 min, or at 4 °C with rotation.
  12. Centrifuge at 420 x g (2,000 rpm) for 5 min at RT, and discard the supernatant.
  13. Wash cells with 1 ml ice cold LBB.
  14. Repeat wash once and discard the supernatant.
  15. Add 100 μl secondary anti-Fc antibody (1:100 in LBB) to all cell pellets, except the unstained cell pellet, and gently vortex to resuspend.
  16. Incubate for 30 min on ice, or at 4 °C with rotation. Cover tubes with aluminum foil to protect the samples from light.
  17. Centrifuge at 420 x g (2,000 rpm) for 5 min at RT, and discard the supernatant.
  18. Wash the cells with 1 ml ice cold LBB.
  19. Repeat wash once and discard supernatant.
  20. Add 250-500 μl of ice cold LBB to each cell pellet, mix and pass through the strainer cap of a 5 ml polystyrene round-bottom tube. This removes clumped cells immediately prior to flow cytometry. It is essential to have a single cell suspension prior to proceeding with flow cytometry–a) Clumped cells interfere with the analysis. b) Clumped cells can clog the flow cytometer.
  21. Proceed to flow cytometry. Care should be taken that samples are exposed to minimum light.

Data analysis

  1. Acquiring data on the flow cytometer
    Unstained cells are used to set the parameters of the flow cytometer. In the flow cytometer acquisition software, open two graph profiles:
    1. Side Scatter (SSC) on the x-axis and Forward Scatter (FSC) on the y-axis.
    2. Histogram on the x-axis and YeFL1 channel on the y-axis (channel on the flow cytometer used to detect the fluorescent secondary antibody).
    3. Set the voltage for SSC vs. FSC such that the majority of the cell population is in the middle of the SSC vs. FSC graph (Figure 2A). Using unstained cell sample, set the second graph histogram vs. YeFL1 channel to a voltage such that the histogram profile is towards the x-axis (≤ 102). Use the same settings to acquire all experimental samples. The samples treated with Fc-tag alone or secondary antibody alone are recorded first and then the samples treated with ligand-Fc are acquired. Acquire at least 20,000 cells per sample for cultured cells. If cell numbers are limiting, the minimum number acquired could be as low as 3,000. Use either the slow or medium speed on the flow cytometer to acquire samples, and keep it the same for all samples. Care should be taken to avoid using the fast run speed. The run speed is determined by the differential pressure applied to move cells through the laser. A high speed increases the number of cells moving through the laser, leading to an increase in coincident events.

  2. Analysis of the data
    Open the entire data set as a workspace on the flow cytometer acquisition software–FlowJo version 10.3.0.Beta3. Other versions of the software can also be used. Using the unstained cells–gate on the mass population of cells using the SSC vs. FSC graph, avoiding small and large or clumped cells. The sub-population of cells gated on will be represented separately, below the main profile of the sample. On this major sub-population of cells change the x-axis to histogram and the y-axis to YeFL1 channel (channel on the flow cytometer used to detect the secondary antibody). Apply this gate to all the samples. The profiles of the Fc alone and Notch ligand-Fc can be plotted using layout editor in the acquisition software (Figure 2B). To compare binding in different sample populations, create overlays in the layout editor. To create overlays: drag the population of the first sample from the workspace to the layout editor, and then drag the second sample population on top of the first graph (Figure 2C). Remember to use the same gate for Fc alone/secondary only and Notch ligand-Fc for each cell type. The mean fluorescence intensity (MFI) of the samples can be calculated by using the statistics toolbar and selecting median on the YeFL1 channel. Data replicates are compared by relative MFI ± SEM; significance determined by paired, two-tailed Student’s t-test n ≥ 3.


    Figure 2. Flow cytometer profile of DLL4-Fc ligand binding to Chinese hamster cells (CHO). A. Profile represents gating on the major population of cells in the SSC vs. FSC profile. B. The profile generated on the gated population of cells treated with Fc-tag alone followed by secondary antibody conjugated to the fluorochrome PE. C. The profile of CHO cells binding Fc-tag alone (dotted line) overlaid with the profile of DLL4-Fc binding (solid line).

Notes

Notch receptor/Notch ligand binding requires the presence of calcium. In the assay above, LBB contains 1 mM CaCl2, binding does not occur in the presence of a low concentration (5 mM) of metal chelator (EDTA or EGTA) (Stahl et al., 2008). This method can be utilized to determine binding parameters for different Notch ligands binding to endogenous or overexpressed Notch receptors present at the surface of any cell type, from cultured to cells isolated from different tissues. This protocol has been tested for adherent cell as well as cells growing in suspension. For example, adherent mouse embryonic stem cells (Stahl et al., 2008) and suspension-grown CHO cells (Hou et al., 2012, Sawaguchi et al., 2016). Cells were always grown at 37 °C in alpha MEM medium containing 10% FBS unless otherwise stated. Ligand binding for each ligand was typically performed in 100 µl of LBB containing 0.5 x 106 cells. Intracellular NECD can be detected by the same method after cell permeabilization. The method is highly sensitive, reliable and reproducible.

Recipes

  1. PBS with cations, pH 7.2-7.4
    137.93 mM sodium chloride
    8.05 mM sodium phosphate dibasic
    1.47 mM potassium phosphate monobasic
    2.67 mM potassium chloride
    0.49 mM magnesium chloride (hexahydrate)
    0.90 mM calcium chloride (anhydride)
  2. Ligand binding buffer (LBB), pH 7.2-7.4
    HBSS
    1% BSA
    1 mM CaCl2
    0.05% sodium azide
  3. 4% PFA in PBS
    Dilute 16% PFA (1 in 4 with PBS containing cations)
  4. Soluble Notch ECD ligand with Fc tag
    1. Briefly, HEK 293T cells stably expressing a construct for Notch ligand with Fc tag (Yang et al., 2005) were grown to 90% confluence before the medium was changed to serum-free Pro293a medium with glutamine
    2. After 72 h, conditioned medium containing secreted Fc-tagged ligand is carefully collected trying not to disturb the cell monolayer. The medium is filtered through a 0.22 µm syringe filter and the filtrate is concentrated using an Amicon Ultra 15 centrifugal unit with 30 Kd molecular weight cutoff
    3. The concentration of the Fc-tagged ligand is determined using Western blot analysis of titrated Notch ligand compared with known amounts of IgG as described (Stahl et al., 2008; Hou et al., 2012)

Acknowledgments

The method described was modified from ‘Roles of Pofut1 and O-fucose in mammalian Notch signaling’ (Stahl et al., 2008), ‘Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced Notch signaling’ (Hou et al., 2012), ‘Lunatic, Manic, and Radical Fringe each promote T and B cell development’ (Song et al., 2016) and ‘O-GlcNAc on Notch1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals’ (Sawaguchi et al., 2017). The authors declare no competing interests. This work was supported by NIGMS RO1 106417 to PS.

References

  1. Benedito, R., Roca, C., Sorensen, I., Adams, S., Gossler, A., Fruttiger, M. and Adams, R. H. (2009). The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137(6): 1124-1135.
  2. Bray, S. J. (2016). Notch signalling in context. Nat Rev Mol Cell Biol 17(11): 722-735.
  3. Bruckner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-delta interactions. Nature 406(6794): 411-415.
  4. Gama-Norton, L., Ferrando, E., Ruiz-Herguido, C., Liu, Z., Guiu, J., Islam, A. B., Lee, S. U., Yan, M., Guidos, C. J., Lopez-Bigas, N., Maeda, T., Espinosa, L., Kopan, R. and Bigas, A. (2015). Notch signal strength controls cell fate in the haemogenic endothelium. Nat Commun 6: 8510.
  5. Haltom, A. R. and Jafar-Nejad, H. (2015). The multiple roles of epidermal growth factor repeat O-glycans in animal development. Glycobiology 25(10): 1027-1042.
  6. Hori, K., Sen, A. and Artavanis-Tsakonas, S. (2013). Notch signaling at a glance. J Cell Sci 126(Pt 10): 2135-2140.
  7. Hou, X., Tashima, Y. and Stanley, P. (2012). Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling. J Biol Chem 287(1): 474-483.
  8. Kopan, R. (2012). Notch signaling. Cold Spring Harb Perspect Biol 4(10).
  9. Kovall, R.A., Gebelein, B., Sprinzak, D., and Kopan, R. (2017). The canonical Notch signaling pathway: Structural and biochemical insights into shape, sugar, and force. Dev Cell 41: 228-241.
  10. Luca, V. C., Jude, K. M., Pierce, N. W., Nachury, M. V., Fischer, S. and Garcia, K. C. (2015). Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science 347(6224): 847-853.
  11. Luca, V. C., Kim, B. C., Ge, C., Kakuda, S., Wu, D., Roein-Peikar, M., Haltiwanger, R. S., Zhu, C., Ha, T. and Garcia, K. C. (2017). Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355(6331): 1320-1324.
  12. Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406(6794): 369-375.
  13. Muller, J., Rana, N. A., Serth, K., Kakuda, S., Haltiwanger, R. S. and Gossler, A. (2014). O-fucosylation of the notch ligand mDLL1 by POFUT1 is dispensable for ligand function. PLoS One 9(2): e88571.
  14. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
  15. Serth, K., Schuster-Gossler, K., Kremmer, E., Hansen, B., Marohn-Kohn, B. and Gossler, A. (2015). O-fucosylation of DLL3 is required for its function during somitogenesis. PLoS One 10(4): e0123776.
  16. Song, Y., Kumar, V., Wei, H. X., Qiu, J. and Stanley, P. (2016). Lunatic, manic, and radical Fringe each promote T and B cell development. J Immunol 196(1): 232-243.
  17. Stanley, P. and Okajima, T. (2010). Roles of glycosylation in Notch signaling. Curr Top Dev Biol 92: 131-164.
  18. Stahl, M., Uemura, K., Ge, C., Shi, S., Tashima, Y. and Stanley, P. (2008). Roles of Pofut1 and O-fucose in mammalian Notch signaling. J Biol Chem 283(20): 13638-13651.
  19. Yang, L. T., Nichols, J. T., Yao, C., Manilay, J. O., Robey, E. A. and Weinmaster, G. (2005). Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1. Mol Biol Cell 16(2): 927-942.

简介

Notch信号传导是进化上保守的信号传导途径,在发育过程中以及在各种组织中维持体内平衡过程中起着不可或缺的作用(Kopan,2012; Hori等人,2013)。 Notch信号的多方面作用在不同的层面上受到严格的调控。监管的最重要的方面之一是不同Notch配体与各Notch受体(NOTCH1-NOTCH4)的结合。典型的配体Delta或Serrate(在果蝇中)和Delta样(DLL1和DLL4)或Jagged(JAG1和JAG2)(在哺乳动物中)是跨膜糖蛋白。在一个细胞上表达的配体结合相邻细胞上的Notch受体以诱导Notch信号传导。通过O-岩藻糖和O-GlcNAc聚糖对Notch受体细胞外结构域的糖基化作为Notch信号强度的关键调节剂已经得到很好的确立(Stanley and Okajima,2010; Haltom and Jafar-Nejad,2015; Sawaguchi et al。 >,2017)。为了表征Notch配体与分离细胞中的Notch受体的结合,我们利用人Fc区在C端标记的Notch配体胞外结构域,并通过流式细胞术确定荧光抗Fc抗体的结合。
【背景】众所周知,细胞增殖,分化和凋亡受Notch信号调节。 Notch信号的异常变化与多种疾病有关,引起一系列发育和成人疾病(Bray,2016)。在哺乳动物中规范的Notch信号传导途径是通过Notch配体Delta或Jagged与Notch受体(NECD)的胞外结构域的结合而启动的,在相反的细胞上表达。受体 - 配体结合启动两个连续的蛋白水解切割,导致Notch胞内结构域(NICD)的释放。与转录阻遏物CSL(CBF-1 /无毛抑制物/ Lag-1)(也称为免疫球蛋白κ区J的重组信号结合蛋白(RBPjk))和共激活物识别物(MAML)缺口目标基因。 Notch受体与不同配体的结合导致不同的结果(Benedito等人,2009; Bray,2016)。例如,造血干细胞的维持受低强度JAG1介导的Notch信号调节,而动脉细胞命运由高强度DLL4介导的Notch信号传导决定(Gama-Norton等人,2015年)。 N-乙酰葡糖胺(GlcNAc)通过穗缘糖基转移酶在NECD的表皮生长因子(EGF)重复上的O-岩藻糖的添加通常增强由δ样配体DLL1和DLL4诱导的Notch受体的信号传导,同时减少由Jagged配体JAG1和JAG2(Bruckner等人,2000; Moloney等人,2000; Yang等人,2005; Kovall等人,等),2017)。最近的结构研究揭示了包括EGF重复序列8-13和可溶性配体DLL4(Luca等人,2015)和JAG1(Luca等人,2015)的Notch1片段上的O-聚糖之间的分子相互作用。,2017)。 Notch配体EGF重复也用O-聚糖修饰,但是缺少O-聚糖的突变配体在很大程度上保持了功能(Muller等人,2014; Serth等人,2015) 。下面描述的方案是确定可溶性Notch配体ECD与内源或引入的Notch受体的ECD的相对结合的方法(图1)。

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图1. Notch配体结合测定的图解。在细胞表面表达的Notch受体具有由29-36个N末端EGF样重复序列组成的胞外域(NECD),随后是3个Lin-12缺口重复序列。使用抗NECD抗体确认细胞表面表达。 NECD非共价连接到胞内域(NICD),其从细胞膜释放,并在配体结合后通过蛋白水解移位到核。 Notch配体Delta样(DLL1和DLL4)和Jagged(JAG1和JAG2)胞外区(ECD)在Notch配体(MNNL)基序的N端包含模块,接着是Delta-Serrate-LAG2(DSL)结构域,随后是6-16个EGF重复序列。对于该测定,Notch配体ECD的C端连接至被荧光标记的二级抗体(PE-Ab)识别的人Fc标签。配体结合缓冲液必须含有钙才能发生Notch配体结合。钙螯合作为配体结合特异性的对照。

关键字:Notch配体结合测定, DLL1, DLL4, JAG1, JAG2, Fc标签, 流式细胞术

材料和试剂

  1. 移液器吸头(USA Scientific,产品目录号:1110-3000,1110-1000,1111-2021)
  2. T75瓶(Corning,Falcon,目录号:353136)或100mm TC-处理的组织培养皿(Corning,Falcon,目录号:353003) >
  3. 15ml Falcon管(Corning,Falcon ,目录号:352099)
  4. 1.5 ml Eppendorf管(USA Scientific,目录号:1615-5500)
  5. 铝箔(Fisher Scientific,目录号:01-213-104)
  6. 带有细胞过滤器盖的5ml聚苯乙烯圆底管(Corning,Falcon,产品目录号:352235)
  7. 注射器衍生的过滤单位,0.22微米(默克,目录号:SLGV013SL)
  8. Amicon离心过滤器,30K(Merck,目录编号:UFC503024)
  9. 无酶细胞解离液(Merck,目录编号:S-014-B)
  10. Alpha MEM培养基(Thermo Fisher Scientific,Gibco TM,产品目录号:11900073)
  11. 胎牛血清(FBS)(Gemini生物产品,目录号:100-106)
  12. Fc受体(FcR)嵌段纯化的大鼠抗小鼠CD16 / CD32,克隆2.4G2(小鼠BD Fc区)(BD,BD Biosciences,目录号:553141)
  13. Notch配体:如先前所述(Stahl等人,2008)从稳定表达配体-Fc构建体的HEK293T细胞产生的在C-末端具有人Fc结构域标签的DLL1,DLL4,JAG1和JAG2 ECD。 。或者,可溶性的Fc-标记的Notch配体可以从R& D Systems:
    购买 DLL1(R& D Systems,目录号:5026-DL-050)
    JAG1(R& D Systems,目录号:599-JG-100)
    JAG2(R& D Systems,目录号:4748-JG-050)
    DLL4(Thermo Fisher Scientific,目录号:10171H02H25)
  14. 第二抗体:R-藻红蛋白(PE)AffiniPure F(ab')2片段山羊抗人IgG,Fcγ片段特异性(Jackson ImmunoResearch,目录号:109-116-170) />
  15. 氯化钠(Fisher Scientific,目录号:S271)
  16. 磷酸氢二钠(Sigma-Aldrich,目录号:S3264)
  17. 磷酸二氢钾(Fisher Scientific,目录号:P285)
  18. 氯化钾(Fisher Scientific,目录号:P217)
  19. 氯化镁六水合物(Fisher Scientific,目录号:M33)
  20. 氯化钙二水合物(Sigma-Aldrich,目录号:C3881)
  21. Hanks平衡盐溶液(HBSS)(Mediatech,目录号:55-022-PB)
  22. 牛血清白蛋白(BSA)(Gemini生物产品,目录号:700-100P)
  23. 叠氮化钠(Fisher Scientific,目录号:BP922I-500)

  24. 在水溶液中的16%多聚甲醛(PFA)(Electron Microscopy Sciences,目录号:15710)
  25. Pro293a TM(Lonza,目录号:12-764Q)
  26. 带阳离子的PBS,pH 7.2-7.4(见食谱)
  27. 配体结合缓冲液(LBB),pH 7.2-7.4(见配方)
  28. 4%的PFA在PBS(见食谱)
  29. 具有Fc标签的可溶性Notch ECD配体(见食谱)

设备

  1. 移液器(Mettler-Toledo,Rainin,目录号:17008653,17008650,17008649; Thermo Fisher Scientific,Thermo Scientific TM,目录号:4641070N)
  2. 台式离心机(GMI,IEC,型号:HN-SII)
  3. 台式离心机(Eppendorf,型号:5417 C)
  4. Coulter粒子计数器(Beckman Coulter,型号:Z1系列)
  5. 流式细胞仪(Cytek Biosciences,型号:DxP 10)

软件

  1. 采集软件:FlowJo CE 7.5.110.7
  2. 分析软件:FlowJo版本10.3.0.Beta3

程序

  1. 对于贴壁细胞
    1. 去除培养基,并在室温(RT)下用5ml冷PBS(阳离子;参见食谱)洗涤细胞层一次。
    2. 删除PBS,并添加1毫升无酶解离试剂每个T75烧瓶或10厘米的碟子解离RT的细胞。
    3. 将烧瓶或培养皿转移至37°C。 1分钟(分钟)后,检查细胞是否已经开始分离。如果不是,将它们保持在37°C直到明显的分离。
    4. 大力挖掘烧瓶或碟子的侧面来解离细胞。
    5. 大部分细胞脱落后,将细胞重新悬浮在9ml含有10%FBS的培养基中以获得单细胞悬液。
    6. 使用无酶解离试剂难以解离的贴壁细胞,可以将细胞从烧瓶或培养皿上刮下,并重新悬浮在含有10%FBS的培养基中的单细胞悬浮液中。小心让聚集的细胞沉降。
  2. 对于上面获得的单细胞悬液和悬浮生长的细胞
    1. 对细胞进行计数 - 如果每个反应使用0.5×10 6个细胞(0.5×10 6个细胞:未染色的细胞来设定,每个重复需要至少2×10 6个细胞/每个配体)流式细胞仪,用于阴性对照的0.5×10 6个细胞,用于测试样品的10 6个细胞[Notch配体-Fc])。
    2. 在台式离心机(GMI,IEC,型号:HN-SII)中,在室温下,将115ml Falcon管中的所需细胞体积以115×g(1,000rpm)离心10分钟。
    3. 吸出上清液并在10ml配体LBB中洗涤细胞沉淀(参见食谱)。小心不要吸出细胞颗粒。
  3. 将细胞固定在4%PFA中
    1. 如上所述离心细胞并吸出上清液。
    2. 每10×7细胞加入1ml 4%PFA到细胞沉淀中,通过在4%PFA中迅速涡旋振荡轻轻地重悬(见食谱)。
    3. 在RT下将细胞在4%PFA中孵育10分钟。
    4. 在115×g(1,000rpm)离心10分钟并弃去上清液。

    5. 用10毫升LBB洗涤细胞两次以上

    6. 在LBB中重悬细胞至终浓度为10 6细胞/毫升
    7. 固定细胞可以在4°C储存至少一个月。使用固定细胞的优点是不会发生膜受体的内吞作用,可以在不同的日子制备不同的细胞类型并固定,并且可以在同一天对每个Notch配体-Fc的所有样品进行配体结合测定,实验,从而减少样品之间的差异。
    8. 未固定的细胞也可以用于Notch配体结合实验。然而,细胞应在指数生长的培养物中于37℃下新鲜使用,并在冷PBS中洗涤,然后在4℃测定结合。为了防止Notch受体的胞吞作用是重要的,并且在该结合测定中可以包含0.05%的叠氮化钠。具有受损质膜的细胞可以使用诸如7-氨基放线菌素D(7-AAD),Hoechst 33342和4,6-二脒基-2-苯基吲哚(DAPI)的染料在LBB中恰在流式细胞术之前加入到细胞中,并随后从细胞门控进行分析。
  4. 实验设计
    标记1.5ml Eppendorf管并根据以下实验设计等分固定数量的细胞。
    1. 实验的控制:
      1. 用于背景荧光的未染色的细胞:将待测定的每种细胞类型的相同数目的混合物等分到1.5ml Eppendorf管中,使得在LBB中的最终细胞数量为0.5-1.0×10 6个细胞。通过涡旋短暂混合。这个样本用于在流式细胞仪中建立参数。
      2. 阴性对照-Fc标记单独或第二抗体单独:取LBB中0.5-1.0×10 6个细胞的等分试样放入1.5ml Eppendorf管中,随后与单独的对照-Fc或二抗一起温育。
        每种细胞类型都设有阴性对照
    2. 测试样品:用Fc标记的不同Notch配体 对于每个Notch配体-Fc,将LBB中的0.5-1.0×10 6个细胞等分到1.5ml的Eppendorf管中。
  5. 在台式离心机(Eppendorf,型号:5417C)中于420℃离心分离上述的420×g(2,000rpm)5分钟,并弃去上清液。
  6. 用1毫升冰冷的LBB洗细胞。
  7. 重复洗一次,丢弃上清。
  8. 除了未受污染的对照细胞(接受50μlLBB)外,向所有细胞中加入50μlFcR阻断剂(在LBB中1:50稀释),并轻轻涡旋。
  9. 在冰上孵育15分钟。不要洗。
  10. 添加100μl单独的Notch配体-Fc或Fc-标签(在100μlLBB中100-1,000ng)。对于未染色的细胞或单独接受二抗的细胞,加入100μlLBB。轻轻地旋转每个管混合。
  11. 在冰上孵育1小时,每隔15分钟手动间歇混合,或者在4°C旋转。
  12. 在室温下,以420×g(2,000rpm)离心5分钟,弃去上清液。
  13. 用1毫升冰冷的LBB洗细胞。
  14. 重复洗一次,丢弃上清。
  15. 除了未染色的细胞沉淀之外,向所有细胞沉淀中加入100μl二抗Fc抗体(LBB中的1:100),轻轻涡旋以重新悬浮。
  16. 在冰上孵育30分钟,或在4°C旋转。用铝箔盖住管子,保护样品免受光照。
  17. 在室温下以420×g(2,000rpm)离心5分钟,弃去上清液。
  18. 用1毫升冰冷的LBB洗细胞。
  19. 重复洗一次,丢弃上清。
  20. 添加250-500微升冰冷LBB到每个细胞沉淀,混合并通过5毫升聚苯乙烯圆底管的过滤器帽。这在流式细胞术之前立即去除聚集的细胞。在进行流式细胞术之前,有必要使用单细胞悬液。a)成团细胞干扰分析。 b)聚集的细胞会堵塞流式细胞仪。
  21. 继续流式细胞术。
    请注意样品暴露在最低限度的光照下

数据分析

  1. 获取流式细胞仪的数据
    未染色的细胞用于设置流式细胞仪的参数。在流式细胞仪采集软件中,打开两个图谱:
    1. X轴上的侧散射(SSC)和Y轴上的前向散射(FSC)。
    2. x轴上的直方图和y轴上的YeFL1通道(用于检测荧光二次抗体的流式细胞仪上的通道)。
    3. 设置SSC对FSC的电压,使得大部分细胞群处于SSC对FSC图的中间(图2A)。使用未染色的细胞样本,将第二个图形直方图与YeFL1通道对应的电压设置为直方图轮廓朝向x轴(≤10 2)。使用相同的设置来获取所有实验样品。首先记录单独用Fc-标签或二次抗体处理的样品,然后获得用配体-Fc处理的样品。每培养细胞每个样品至少获得20,000个细胞。如果手机号码是有限的,获得的最低数量可能低至3000。使用流式细胞仪上的慢速或中速采集样本,并保持所有样本相同。应该小心避免使用快速运行速度。运行速度取决于通过激光器移动细胞所施加的压差。高速度增加了通过激光器的细胞数量,导致同时发生的事件增多。

  2. 分析数据
    在流式细胞仪采集软件FlowJo 10.3.0.Beta3上打开整个数据集作为工作区。其他版本的软件也可以使用。使用SSC对FSC图,在大量细胞群上使用未染色的细胞门,避免小的或大的或成团的细胞。门控细胞的亚群将在样本的主要概况下单独表示。在这个主要细胞亚群中,将x轴改变为直方图,将y轴改变为YeFL1通道(用于检测二抗的流式细胞仪上的通道)。将这个门应用到所有的样本。可以使用采集软件中的布局编辑器绘制单独的Fc和Notch配体-Fc的图谱(图2B)。要比较不同样本群体中的结合,请在布局编辑器中创建叠加层。要创建叠加:将第一个样本的填充从工作空间拖放到布局编辑器,然后将第二个样本填充到第一个图的顶部(图2C)。请记住,对于每个细胞类型,仅使用与单独的Fc /次级相同的门和Notch配体-Fc。样品的平均荧光强度(MFI)可通过使用统计工具栏并在YeFL1通道上选择中位数来计算。通过相对MFI±SEM比较数据重复;意义由成对的双尾学生的测验确定n = 3。


    图2.与中国仓鼠细胞(CHO)结合的DLL4-Fc配体的流式细胞仪概况。 :一种。配置文件代表SSC与FSC配置文件中主要细胞群的门控。 B.在仅用Fc-标签处理的门控细胞群体上产生的图谱,然后是与荧光染料PE缀合的第二抗体。 C.单独结合Fc-标签的CHO细胞的概况(虚线)覆盖了DLL4-Fc结合(实线)的图谱。

笔记

Notch受体/ Notch配体结合需要钙的存在。在上述测定中,LBB含有1mM CaCl 2 2,在低浓度(5mM)金属螯合剂(EDTA或EGTA)存在下不发生结合(Stahl等, ,2008)。该方法可用于确定不同Notch配体与任何细胞类型表面上存在的内源性或过表达Notch受体结合的结合参数,从培养的细胞分离到不同组织。此协议已经测试了贴壁细胞以及悬浮生长的细胞。例如,贴壁的小鼠胚胎干细胞(Stahl等人,2008)和悬浮生长的CHO细胞(Hou等人,2012,Sawaguchi等人。,2016)。除非另有说明,细胞始终在含有10%FBS的αMEM培养基中于37℃生长。通常在100μl含有0.5×10 6个细胞的LBB中进行每个配体的配体结合。细胞透化后可以用相同的方法检测细胞内NECD。该方法灵敏度高,可靠,重现性好。

食谱

  1. 带有阳离子的PBS,pH 7.2-7.4
    137.93 mM氯化钠
    8.05 mM磷酸二氢钠
    1.47 mM磷酸二氢钾
    2.67mM氯化钾
    0.49mM氯化镁(六水合物)
    0.90 mM氯化钙(酸酐)
  2. 配体结合缓冲液(LBB),pH 7.2-7.4
    HBSS
    1%BSA
    1 mM CaCl 2 2/2 0.05%叠氮钠
  3. PBS中4%PFA
    稀释16%的PFA(1/4含有阳离子的PBS)
  4. 带有Fc标签的可溶性Notch ECD配体
    1. 简言之,将稳定表达具有Fc标记的Notch配体的构建体(Yang等,2005)的HEK 293T细胞生长至90%铺满,然后将培养基更换为无谷氨酰胺的无血清Pro293a培养基br />
    2. 72小时后,仔细收集含有分泌的Fc-标记的配体的条件培养基,试图不干扰细胞单层。将培养基通过0.22μm注射器过滤器过滤,并使用具有30Kd分子量截留的Amicon Ultra 15离心单元浓缩滤液。
    3. 如已经描述的(Stahl等人,2008; Hou等人,J.Biol.Chem。 em>,2012)

致谢

所描述的方法从“Pofut1和O-岩藻糖在哺乳动物Notch信号传导中的作用”(Stahl等人,2008),“半乳糖差异调节对Delta1诱导的Notch信号传导的疯狂和狂躁边缘效应(Hou等人,2012),“疯狂,疯狂和自由基边缘各自促进T和B细胞发育”(Song等人,2016)和“ Notch1上的O-GlcNAc EGF重复序列调节哺乳动物中配体诱导的Notch信号传导和血管发育(Sawaguchi等人,2017)。作者声明没有竞争的利益。这项工作得到了NIGMS RO1 106417对PS的支持。

参考

  1. Benedito,R.,Roca,C.,Sorensen,I.,Adams,S.,Gossler,A.,Fruttiger,M.和Adams,R.H。(2009)。缺口配体Dll4和Jagged1对血管生成具有相反的作用。 Cell 137(6):1124-1135。
  2. Bray,S.J。(2016)。上下文中的Notch信令。 Nat Rev Mol Cell Biol 17(11):722-735。
  3. Bruckner,K.,Perez,L.,Clausen,H.和Cohen,S。(2000)。边缘的糖基转移酶活性调节Notch-δ相互作用。 Nature 406(6794):411-415。
  4. Gama-Norton,L.,Ferrando,E.,Ruiz-Herguido,C.,Liu,Z.,Guiu,J.,Islam,AB,Lee,SU,Yan,M.,Guidos,CJ,Lopez-Bigas, N.,Maeda,T.,Espinosa,L.,Kopan,R.和Bigas,A。(2015)。 Notch信号强度控制血管内皮细胞的命运。 Nat Commun 6:8510.
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Copyright Varshney and Stanley. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Varshney, S. and Stanley, P. (2017). Notch Ligand Binding Assay Using Flow Cytometry. Bio-protocol 7(23): e2637. DOI: 10.21769/BioProtoc.2637.
  2. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
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