Notch Ligand Binding Assay Using Flow Cytometry

Notch signaling is an evolutionary 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.


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. Receptorligand 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 This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). example, the maintenance of hematopoietic stem cells is regulated by low strength JAG1mediated Notch signaling, whereas arterial cell fate is determined by high strength DLL4mediated 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 Oglycans 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

a.
Remove culture medium and wash the cell layer once with 5 ml of cold PBS (with cations;see Recipes) at room temperature (RT).

b.
Remove PBS and add 1 ml of enzyme-free dissociation reagent per T75 flask or 10 cm dish to dissociate the cells at RT.

c.
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.

d.
Vigorously tap the sides of the flask or dish to dissociate the cells.

e.
After most cells have detached, re-suspend the cells in 9 ml of medium containing 10% FBS to obtain a single cell suspension.

f.
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 a.

b.
Centrifuge the required cell volume in a 15 ml Falcon tube at 115 × g (1,000 rpm) for 10 min in a benchtop centrifuge (GMI, IEC, model: HN-SII) at RT.

c.
Aspirate the supernatant and wash the cell pellet in 10 ml ligand LBB (see Recipes). Be careful not to aspirate the cell pellet.

a.
Centrifuge cells as above and aspirate supernatant.

b.
Add 1 ml of 4% PFA per 10 7 cells to the cell pellet and gently resuspend by vortexing in brief spurts in 4% PFA (see Recipes).

c.
Incubate cells in 4% PFA for 10 min at RT.

e.
Wash the cells twice more with 10 ml LBB.

f.
Resuspend cells to a final concentration of 10 6 cells/ml in LBB.

g.
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.

h.
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.

a.
a. Controls for the experiment: i.
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 × 10 6 cells in LBB. Mix briefly by vortexing. This sample is used to establish parameters in the flow cytometer.

ii.
Negative controls-Fc tag alone or secondary antibody alone: Take an aliquot with 0.5-1.0 × 10 6 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.

b. Test samples: Different Notch ligands tagged with Fc
For each Notch ligand-Fc, aliquot 0.5-1.0 × 10 6 cells in LBB into separate 1.5 ml Eppendorf tubes.

7.
Repeat the wash once and discard the supernatant.

9.
Incubate for 15 min on ice. Do not wash.

11.
Incubate for 1 h on ice with intermittent mixing by hand every 15 min, or at 4 °C with rotation.

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.

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.

A.
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 yaxis.

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 xaxis (≤ 10 2 ). 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.

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 subpopulation 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.

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 × 10 6 cells. Intracellular NECD can be detected by the same method after cell permeabilization. The method is highly sensitive, reliable and reproducible.  (Yang et al., 2005) were grown to 90% confluence before the medium was changed to serum-free Pro293a medium with glutamine

b.
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 c.
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)  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 Deltalike (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 fluorescentlylabeled 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. Bio Protoc. Author manuscript; available in PMC 2018 January 10.