Notch Ligand Binding Assay Using Flow Cytometry

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, Gibco^TM, 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. Pro293a^TM (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 Scientific^TM, 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.

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