Protocol to study juxtacrine signaling in human vascular cells using a fluorescence labeling co-culture approach

Summary

Endothelial cells interact closely with adjacent endothelial cells and pericytes. The intercellular crosstalk between these cells is essential for angiogenesis, maturation, and vascular homeostasis. This protocol outlines a method to study these interactions using a co-culture system. We describe steps for transducing/transfecting, labeling, and co-culturing human vascular endothelial cells and pericytes. We then detail procedures for immunofluorescence staining and microscopy imaging. This protocol enables molecular in vitro investigations of juxtacrine signaling pathways and gene expression.

For complete details on the use and execution of this protocol, please refer to Herrera et al.¹

Graphical abstract

Highlights

• •Instructions for the expansion and transduction of primary vascular cells
• •Guidance on how to use the fluorescent probes effectively
• •Steps for labeling, co-culturing, immunostaining, and imaging cells
• •Procedure to analyze the co-culture by flow cytometry

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Endothelial cells interact closely with adjacent endothelial cells and pericytes. The intercellular crosstalk between these cells is essential for angiogenesis, maturation, and vascular homeostasis. This protocol outlines a method to study these interactions using a co-culture system. We describe steps for transducing/transfecting, labeling, and co-culturing human vascular endothelial cells and pericytes. We then detail procedures for immunofluorescence staining and microscopy imaging. This protocol enables molecular in vitro investigations of juxtacrine signaling pathways and gene expression.

Before you begin

This protocol describes the generation of fluorescently labeled vascular cell coculture systems to analyze juxtacrine signaling between these cells. It is associated with the research paper Herrera & Komatsu (2024) in which we employed the coculture method to demonstrate that Akt3 activation by R-Ras in a quiescent endothelial cell (EC) guides the quiescence and barrier stability of neighboring ECs via Jagged1-Notch signaling.¹

We will first describe a protocol for studying juxtacrine signaling in a coculture of wild-type ECs and ECs overexpressing a gene of interest. The effect of the loss-of-function of a gene can be studied in a similar manner using a coculture of wild-type ECs and gene-silenced ECs. We use lentivirus-mediated cDNA transduction or siRNA-transfection of ECs cocultured with control ECs for this purpose. The protocol includes descriptions of optimal seeding ratios for analyzing intercellular crosstalk such as the Jagged1-Notch signaling between neighboring ECs. This protocol also covers the method to study the crosstalk between ECs and pericytes. Next, we will describe the detailed methods for immunofluorescence staining of the coculture and the subsequent image acquisition. Thirdly, we will describe a protocol combining these methods with cell sorting using Flow Cytometry. This strategy permits us to investigate specific vascular cell populations for further biochemical, molecular biological, and genomic analyses. Examples of additional applications include analyses of proliferation or migration of either one of the two cell types in the coculture and analyses of molecular interactions between the two cell types. In 3-D coculture using Matrigel, fluorescently labeled cells are useful for studying interactions between EC-EC and EC-PC during microvessel assembly.² These methods can also be adapted to the studies of interactions between other cell types, such as vascular smooth muscle cells, fibroblasts, immune cells, and cancer cells. For example, cocultures can be used for modeling the tumor microenvironment in which cancer cells and leukocytes are labeled with different fluorophores to study molecular interactions between them.³ Likewise, the interaction between leukocyte and EC monolayer in coculture provides an in vitro model of leukocyte adhesion and extravasation processes.⁴ A schematic workflow of these protocols is provided in Figure 1.

Figure 1.

Workflow and options for coculture of genetically engineered vascular cells

Institutional permissions

The protocols included in this manuscript utilized commercially available cells and reagents. The protocol can be readily adapted to the studies using primary cells isolated from various animal models and clinical samples. Nonetheless, readers are reminded to follow all appropriate institutional requirements for the use of such materials.

Lentiviral transduction of endothelial cells and stock generation

Timing: 7 days

Human umbilical vein endothelial cells (HUVEC) from pooled donors and mixed sexes (purchased from Lonza) are used at passage 1–6. These cells are transduced with a lentiviral vector carrying cDNA for constitutively active small GTPase R-Ras (R-Ras38V) or an insertless vector (mock).⁵ Human brain microvascular pericytes (PC, purchased from ScienCell) are used for studying EC-PC interaction.

• 1.Thaw a vial of parental endothelial cells (HUVEC) by placing it in a water bath at 37°C for 1 min, and transfer the content immediately to a 15-mL conical tube containing 4 mL of freshly-made cell growth medium (see key resources table) in a safety cabinet. Centrifuge at 0.4 rcf for 5 min. Aspirate carefully and resuspend the pellet with 10 mL of cell growth medium, and plate the cells on a 10 cm dish. Incubate them at 5% CO₂, 37°C until 80% confluency.
• 2.
  Split cells to expand and transduce ECs to generate a stock:

  • a.
    Aspirate cell medium and wash cells twice with 5 mL of PBS at room temperature.
  • b.
    Add 3 mL of pre-heated 0.25% trypsin-EDTA and incubate the culture dish at 37°C for 2–4 min.
  • c.
    Check cells on a microscope to observe that cells have detached.
  • d.
    Add 3 mL of appropriate pre-heated cell medium.
  • e.
    Transfer cell suspension to a 15 mL conical tube and centrifuge at 0.4 rcf for 5 min.
  • f.
    Gently aspirate the supernatant and carefully resuspend the pellet in 2 mL cell medium.
  • g.
    Take a 10 μL aliquot of the cell suspension and mix it up with 10 μL of Trypan blue.
  • h.
    Check viability by a cell counter.
  • i.
    Seed 5×10⁵ cells per 10 cm dish.
  • j.
    24–30 h later, transduce EC with mock or R-Ras38V lentiviruses with a 0.5 MOI in 10 mL of cell medium.

    Note: This protocol does not include steps for lentivirus titration. Users must know the Transduction Units (TU) of their lentiviral stocks. Please, refer to Iggo (2022) for a protocol for titration.⁶

    CRITICAL: HUVECs have a doubling time of approximately 24–30 hours. Expect to have around 1×10⁶ cells on each dish by this time, right before starting the transduction. Consider this to accurately estimate the correct amount of Transduction Units of virus to use per dish based on the desired MOI.

    CRITICAL: All experiments involving lentiviral particles must be performed in a biosafety level 2 (BSL2) laboratory using appropriate Personal Protective Equipment (PPE).

  • k.
    Add polybrene (hexadimethrine bromide) to a final concentration of 10 μg/mL to each dish to increase the efficiency of infection.
  • l.
    After 14–16 h, wash cells twice with PBS and replace with 10 mL of cell medium.
  • m.
    Wait for at least 3 days or until cells reach confluency.
• 3.
  Split transduced EC to generate a stock:

  • a.
    Label cryovials.
  • b.
    Repeat steps 2a through 2h.
  • c.
    Dilute your cell suspension in complete cell medium to a final concentration of 5×10⁵ cells/mL containing 10% DMSO.
  • d.
    Make 1 mL aliquots for mock or R-Ras38V-transduced ECs using cryovials.
  • e.
    Place cryovials in a cryo-safe freezing container and transfer it immediately to an −80°C freezer. Store them for at least one day and up to one week.
  • f.
    1–7 days later, transfer cryovials containing the transduced cells to a liquid nitrogen tank.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal anti-p21	Cell Signaling Technology	Cat #2947; RRID:AB_823586
Rabbit polyclonal anti-Hey1	GeneTex	GTX118007; RRID:AB_11168085
Mouse monoclonal anti-human VE-cadherin	Santa Cruz	sc-9989; RRID:AB_2077957
Goat polyclonal anti-mouse Alexa Fluor-647	Thermo Fisher Scientific	A21236
Goat polyclonal anti-rabbit Alexa Fluor-555	Thermo Fisher Scientific	A21429
Bacterial and virus strains
Insertless control pLenti6/V5 lentivirus expression vector and R-Ras38V.	Thermo Fisher Scientific	Komatsu & Ruoslahti5
Chemicals, peptides, and recombinant proteins
CellTracker Green CMFDA	Invitrogen	C7025
CellTracker Red CMTPX	Invitrogen	C34552
Dimethyl sulfoxide (DMSO)	Fisher Bioreagents	BP231-100
0.25% trypsin-EDTA (1X)	Gibco	25200-056
Endothelial cell growth base media (EBM)	R&D Systems	390598
Endothelial cell growth supplement (EGM)	R&D Systems	390599
Dulbecco’s phosphate-buffered saline (DPBS)	Corning	21-031-CV
Pericyte medium (PM)	ScienCell	1201
Endothelial cell growth basal medium phenol red free	Lonza	CC-3129
Mojo sort buffer (FACS)	BioLegend	480017
4-well chamber glass slide with cover	Lab-Tek	154526
Cytoseal 60	Epredia	8310-4
Fluoromount-G	SouthernBiotech	0100-01
4′,6-diamidino-2-phenylindole (DAPI)	Tocris	5748
eFluor 780 viability dye	Invitrogen	65-0865-14
5 mL polystyrene round-bottom tube (FACS)	Falcon	352235
Experimental models: Cell lines
Human umbilical vein cells (HUVECs)	Lonza	C2519A
Human brain vascular pericytes (HBVPCs)	ScienCell	1200
Software and algorithms
NIS-Elements Advanced Research 5.21.03	Nikon	https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research
Other
Centrifuge to pellet primary cells	Eppendorf 5702

Step-by-step method details

Co-culture of vascular cells

Timing: 7–8 days

This protocol describes the coculture of genetically modified human endothelial cells. This protocol has three steps: the expansion of primary vascular cells, the labeling with fluorescent intracellular dyes, and the coculture set up.

Culture of primary cells

Timing: 7–8 days

• 1.Prepare fresh cell culture medium (KRT). Incubate cells at 5% CO₂, 37°C until confluency.
• 2.
  Prepare cultures of EC and PC as follows:

  • a.
    Day 1: Thaw R-Ras38V or mock transduced-ECs and parental ECs from liquid nitrogen tank as described in Lentiviral transduction of endothelial cells and stock generation.
  • b.
    Culture these cells in separate 10 cm dishes using 12 mL of appropriate culture medium.

    CRITICAL: All experiments involving lentiviral particles must be performed in a biosafety level 2 (BSL2) laboratory.

  • c.
    Day 2: Check cell quality to make sure that the cells are suitable for coculture. HUVECs have a doubling time of approximately 24–30 h, and they exhibit a characteristic morphology of cultured ECs (Figure 2A). Change medium to remove unattached cells.

    Note: For a healthy and successful coculture, always start expansion of cells from low passages (1 or 2). Both transduced- and non-transduced EC should be the same passage for setting up a coculture.

  • d.
    Day 3: For EC-PC coculture, thaw pericytes from liquid nitrogen tank. Culture these cells in a 10 cm dish using 12 mL of appropriate culture medium. PCs have a doubling time of approximately 12–15 h.

    Note: For consistent, reproducible results, PCs should be used at low passages similar to ECs.

  • e.
    Day 4: Check PC culture and replace medium with fresh growth medium. Allow ECs to grow at least a passage prior to use in coculture. This permits cell expansion into different dishes for each experimental condition to study.

    • i.
      Aspirate culture medium and wash with 5 mL PBS twice.
    • ii.
      Add 3 mL of 0.25% trypsin-EDTA and incubate plates at 37°C for 3–5 min.
    • iii.
      When cells are detached, add 3 mL of medium and transfer cell suspension to 15 mL conical tubes for centrifugation at 0.4 rcf for 5 min.

      Note: Split EC or PC when they reach 80–90% confluency.

  • f.
    Day 5: Allow PCs to grow at least once prior to use in coculture.
  • g.
    Day 7: Check EC and PC confluency.

    • i.
      If no further genetic manipulation by siRNA is desired, proceed to step 2 to label the vascular cells with fluorescence dyes.
    • ii.
      If siRNA knockdown is to be performed in any of the cell types, proceed with overnight siRNA transfection (not included in this protocol) and perform the fluorescence cell labeling next day. Refer to the troubleshooting section for tips.

      CRITICAL: Make sure the cells reach 80–90% confluency before proceeding to cell labeling.

Figure 2.

Labeling of vascular cells

(A) HUVEC cells at confluency. Scale bar 500 μm; (B) Culture dish of the cells labeled with 5 μM Green CMFDA Cell Tracker (left) or 3 μM Red CMTPX Cell Tracker (right) at time zero; (C) Culture dish at 45 min of incubation (D) 3-D Matrigel coculture of EC (white) and PC (red) imaged by confocal microscopy.

Fluorescence cell labeling

Timing: 1 h

• 3.
  Reconstitute green-fluorescence (CellTracker Green CMFDA) or red-fluorescence (CellTracker Red CMTPX) dyes in sterile DMSO in a biosafety cabinet at room temperature to a final concentration of 10 mM.

  • a.
    Gently vortex the vial to help dissolve the content.
  • b.
    Prepare the final working solution in pre-warmed (37°C) serum-free, phenol red-free basal medium in 15 mL conical tubes.

CRITICAL: CellTracker dyes are light sensitive. Turn off the biosafety cabinet light and cover the tubes containing the working solution with aluminum foil. They are also sensitive to freeze-thaw cycles. Use freshly prepared dyes only.

• 4.
  Cell labeling.

  • a.
    Remove cell medium from culture dish by gentle aspiration.
  • b.
    Wash cells twice with sterile PBS.
  • c.
    Add the working solution of fluorescence dye to each dish as follows:

    • i.
      5 μM Green CMFDA solution for transduced and/or siRNA-transfected ECs.
    • ii.
      Phenol red-free basal medium to non-transduced ECs.
    • iii.
      3 μM Red CMTPX solution for PCs (in case of EC-PC coculture).
  • d.
    Incubate for 45 min at 37°C in the CO₂ incubator (Figure 2B).
  • e.
    After incubation (Figure 2C), wash cells twice with PBS, add cell growth medium (phenol red-free) and incubate overnight.
  • f.
    The day after, corroborate that cells look healthy using a bright-field microscope.

Note: Green CMFDA Cell Tracker solution turns to green color after 10–15 min of incubation. Red CMTPX Cell Tracker solution is originally bluish color, and it turns to purple overtime (Figures 2B and 2C).

CRITICAL: The final concentration of Cell Tracker dyes can be toxic to cells depending on the cell type and time of incubation. The concentration and incubation time for EC and PC provided in this protocol has been optimized. It is safe and produces nontoxic effects on these cells, and it is brightly fluorescent and durable for 4–5 days. Do not overload cells with Cell Tracker dyes. It is crucial to check that cells look healthy throughout the labeling process. Refer to the troubleshooting section if you detect unhealthy cells.

Coculture setup

Timing: 1 h 30 min

• 5.
  Set up the co-culture.

  • a.
    Remove cell medium from culture dish by gentle aspiration.
  • b.
    Wash cells twice with sterile PBS.
  • c.
    Add 3 mL of 0.25% trypsin-EDTA and incubate plates at 37°C for 3–5 min.
  • d.
    When cells are detached, add 3 mL of medium and transfer cell suspension to 15 mL conical tubes for centrifugation at 0.4 rcf for 4 min.
  • e.
    Resuspend the cell pellet with 3 mL growth medium by slowly pipetting up and down 4–5 times.
  • f.
    Determine cell number and viability using a cell counter.
• 6.For EC-EC coculture, plate fluorescently labeled ECs with unlabeled ECs at [1:3] ratio into a 4-well chamber slide. Culture for 48–72 h.
• 7.For EC-PC coculture, plate fluorescently labeled EC with PC at a 1:10 ratio into a 4-well chamber slide. Pericytes must be resuspended in PC growth medium, and after cell counting, an aliquot containing the number of PCs of interest must be added to the EC seeded in EC complete growth media. Thus, the co-culture is maintained using EC growth medium. Incubate for 48–72 h.

CRITICAL: Make sure the cells reach 90% confluency before proceeding with immunostaining of your coculture as juxtacrine signaling requires direct cell surface contact between adjacent cells. Refer to the troubleshooting section for more details.

Alternatives: Other seeding ratios of each cell type can be used depending on the study aim.

Alternatives: Although not described in this protocol, the EC-PC coculture can also be set up in 3-D Matrigel to study microvessel formation in vitro. Find an example in Figure 2D.

Note: If performing a coculture assay with chemical or drug treatment (e.g. neutralizing antibodies, γ-secretase inhibitors to block Notch signaling, cell contraction inhibitors, agonist, etc.), add the drug or compound during step 6 or 7 at the desired concentration to the culture medium. Repeat if necessary, according to the drug properties.

Immunostaining and image acquisition

Timing: 24 h

This protocol describes two steps: immunostaining of coculture with desired antibodies and image acquisition using NIS-Elements imaging software (Nikon Instruments Inc.).

Immunofluorescence staining

Timing: 24 h

CRITICAL: Perform all staining steps in cell chambers protected from light as much as possible. Cell Tracker dyes are light sensitive. Keep your chambers covered with aluminum foil during washing steps.

CRITICAL: Perform all staining steps with gentle agitation to ensure homogenous washing, fixation, permeabilization, blocking, and antibody incubation.

• 8.Carefully aspirate cell medium from 4-well chambers and wash three times with PBS.
• 9.Fix cells with freshly prepared 4% formaldehyde for 15 min at room temperature in a fume hood using the appropriate PPE. Discard according to the institutional guidelines.
• 10.Permeabilize cells with freshly prepared 0.1% Triton X-100-PBS solution for 15 min.
• 11.Block with freshly prepared and filtered 1% BSA-PBS solution for 1 h at room temperature. No shaking needed.
• 12.Incubate overnight with primary antibodies of interest at optimized concentration at 4°C (key resources table).
• 13.Next day, wash cells 3 times with PBS for 5 min.
• 14.Incubate with Alexa-conjugated secondary antibodies for 45 min (key resources table).
• 15.Wash cells 3 times in PBS for 5 min.
• 16.Incubate with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 10 min.
• 17.Wash cells 2 times with PBS and mount slides with 75 μL Fluoromount-G or Cytoseal 60.

Note: Skip the cell permeabilization step if no intracellular target proteins will be analyzed.

Note: Make sure to filter the Blocking solution using a 0.45-μm filter.

Note: When using primary antibodies conjugated with fluorophores, skip Step 14 and 15.

Note: Water-based or toluene-based mounting medium can be used.

Microscopy imaging

• 18.Place your slide in the imaging system.
• 19.Focus your cells using the laser 405 (DAPI) to avoid bleaching the green and red dyes. We used a Nikon A1R Confocal Microscope equipped with a resonant disc (Figure 2C) or a Nikon Eclipse 90i upright fluorescence microscope (Figure 3). For details about the instruments’ metadata and the image acquisition attributes, please refer to Figure S1 (S1; Nikon A1R Confocal Microscope) or to Figures S2 and S3 (S2; Nikon Eclipse 90i microscope).

CRITICAL: Cell Tracker dyes can bleach quickly. Minimize the exposure time (fluorescence microscope) or the gain and voltage (confocal microscope) in the channels used for each fluorescence dye.

• 20.Adjust fluorescence intensity for all channels separately, avoiding signal saturation.

CRITICAL: The saturation of fluorescence intensity impedes the accurate quantification of the fluorescence signal. Make sure you are at least one third below the maximum intensity of the dynamic range of your camera.

• 21.If necessary, obtain a Z-stack of multiple plane images.
• 22.Scan and save your image files.

Figure 3.

EC-EC and EC-PC cocultures

(A) Coculture of transduced EC (green) and non-transduced EC (DAPI only) immunostained for the Notch target gene Hey1 (magenta); Scale bar: 50 μm (B) Coculture of PC (green) and EC subsequently immunostained for p21 (red); Scale bar: 20 μm.

Coculture flow cytometry

Fluorescently labeled cells in coculture can be isolated by flow cytometry (Figure 4), which allows us to separately analyze each cell type in subsequent assays.

CRITICAL: This protocol describes flow cytometry sorting using the BD FACS Aria Ilu Flow Cell Sorter (BD Biosciences) with a flow rate of 1K events/second. The procedure has also been successfully performed using CytoFlex LX (Beckman Coulter). The optimal flow rates and laser settings may differ depending on cell sorters.

Alternatives: EC-EC coculture can also be labeled with 2 colors and use flow cytometry to segregate the two populations of endothelial cells.

Figure 4.

FACS analysis of EC-PC coculture

The pseudocolor plots show the gating strategy to determine the ratio of EC:PC.

Flow cytometry

Timing: 24 h

• 23.Refer to Step 2 sub-step 4 for fluorescence labeling of cells.
• 24.Set up the FACS sorter to detect green CMFDA-labeled ECs, red CMTPX-labeled PC, and dead cells labeled with the cell viability dye eFluor 780. Green.

Note: Green fluorescence from CMFDA dye can be detected using the FITC (fluorescein isothiocyanate) channel (excitation wavelength 488 nm; detection wavelength 530 nm). Red fluorescence from CMTPX dye can be detected using the PE (phycoerythrin) channel (excitation wavelength 561 nm; detection wavelength 585 nm). eFluor 780 fluorescence can be detected in the APC-Cy7 (allophycocyanin-cyanine 7) channel (excitation wavelength 650 nm; detection wavelength 780 nm).

• 25.Set up a gating strategy that includes the detection of single color-stained control cells.
• 26.Remove cell medium from culture dish by gentle aspiration.
• 27.Wash cells twice with 1X Dulbecco’s Phosphate-buffered saline (DPBS).
• 28.Add 3 mL of 0.25% trypsin-EDTA and incubate plates at 37°C for 3–5 min.
• 29.When cells are detached, add 3 mL of medium and transfer cell suspension to 15 mL conical tubes for centrifugation at 0.4 rcf for 4 min.
• 30.Carefully aspirate the supernatant and resuspend the pellets in 1 mL PBS containing 1 μL of cell viability dye eFluor 780 (APC-Cy7-A) for 10 min.
• 31.Centrifuge at 0.4 rcf for 4 min and resuspend in 1 mL 1x Mojo Sort Buffer.
• 32.Filter the cell suspension using a 5 mL polystyrene round-bottom tube with 40-μm cell-strainer cap.

CRITICAL: Not filtering the cell suspension may clog in the flow cytometer. Sub-step 32 is critical to generate a single cell suspension and to prevent sorting of doublets.

• 33.Gate the cell population using forward scatter (FSC-A) and side scatter (SSC-A). Refer to the troubleshooting section if events do not display on the screen.
• 34.Select singlet using FSC-A and FSC-H on the density plots.
• 35.Gate the eFluor 780 negative population to select the live cells. Exclude dead cells with higher autofluorescence from the analysis.
• 36.Detect and sort the Green CMFDA-labeled ECs and the Red CMTPX-labeled PCs. Sort ECs into tubes containing 200 μL EGM media and PCs into PM media (KRT).

Expected outcomes

Expected outcomes: Immunofluorescence

The example illustrated in Figure 3A shows a coculture of fluorescently labeled R-Ras38V-transduced ECs with non-transduced unlabeled ECs, followed by immunostaining with anti-Hey1 antibody. This study demonstrated that R-Ras activation in ECs induces Notch target gene (Hey1) in neighboring ECs, enforcing quiescence of these cells.

The example illustrated in Figure 3B shows a coculture of fluorescently labeled R-Ras38V-transduced ECs with non-transduced fluorescently labeled PCs, followed by immunostaining with anti-p21 antibody. This study demonstrated that R-Ras activation in ECs suppresses proliferation of PCs that are directly interacting with these ECs.

Expected outcomes: Flow cytometry

The example illustrated in Figure 4 shows the separation of co-cultured cell populations by FACS sorting. The use of fluorescence cell labeling in coculture enables isolation of individual cell populations after the coculture, which can be further processed for subsequent applications such as RNA and protein extractions to conduct transcriptomics and biochemical analyses for each cell type. Flow Cytometry can also determine the relative abundance of fluorescently labeled and unlabeled cells at the end of coculture to assess the effect of gene expression on cell proliferation. In conjunction with image analyses by microscopy, it is possible to conduct detailed analyses of signal transduction pathways.

Limitations

The silencing of certain genes can negatively affect the cell viability or growth making it difficult to produce a confluent monolayer necessary for the evaluation of juxtacrine signaling in the coculture. This is exemplified in Figure 5, where silencing AKT1, AKT2, NOTCH1, or NOTCH3 is detrimental to EC proliferation and survival (Figures 5A and 5B). Compared with parental cells, genetically engineered vascular cells may be more sensitive to several manipulation steps required for the coculture setup.

Figure 5.

Troubleshooting

(A) Effect of siAkt1 or siAkt2 knockdown on ECs 72 h post-transfection. These cells failed to make confluent monolayer (B) Effect of siNotch1 or siNotch3 on ECs 72 h post-transfection. These cells failed to make confluent monolayer.

(C) EC-EC coculture at different confluency stained for VE-cadherin (magenta). Scale bar: 100 μm.

Troubleshooting

Problem 1

Poor cell survival after siRNA or shRNA silencing of the gene of interest (Step 1, sub-steps g, II; Figures 5A and 5B).

Potential solution

• •Perform reverse siRNA transfection in the cell type whose gene is to be silenced, prior to adding the second cell type. This omits many washing steps and poses less stress for cells.
• •Use specific inhibitors, for instance γ-secretase inhibitors for inhibiting Notch signaling, instead of gene silencing. If inhibitors or agonists were to be used for the coculture study, it is essential to design the method in a way that only one of the two cell types is affected in the coculture by this treatment. Therefore, the target cell type must be pretreated with the inhibitor/agonist prior to the coculture setup.

Problem 2

Cells do not look healthy after cell labeling with CellTracker. Many cells are dead and/or floating.

Potential solution

• •Use a lower concentration of CellTracker.
• •Incubate for less time (20–30 min).
• •After labeling, wash cells three times with PBS, 30 s each time, to make sure that all unbound dye is removed from the cell medium.

Problem 3

No juxtacrine signaling or weak immunostaining of adherens junctions (VE-cadherin) detected (sub step 20-22; Figure 5C).

Potential solution

• •Make sure your coculture is sufficiently confluent, at least 90%. Notch and other juxtacrine pathways require direct cell surface contact between neighboring cells to transduce signals (Figure 5C).
• •Keep the coculture for at least 72 h. Give cells time to interact, to upregulate junctional proteins and to translocate them to the plasma membrane.
• •Using PBS containing Ca²⁺ for washing after the addition of DAPI may preserve adherens junction interactions prior to fixation. All cadherins contain calcium-binding domains required for calcium-dependent homophilic adhesion between cells. The use of Ca²⁺ containing PBS fortifies adherens junctions during cell washing process and improves the staining pattern.

Problem 4

No events detected in the flow cytometry analysis (sub-step 33-36).

Potential solution

• •First, rule out any technical issues with the operation of the flow cytometer such as air in the system or laser/detector failure. If you do not have such issues, it is most likely that you need to decrease the gain for the channels where you expect to see the Green CMFDA⁺ population (EC) or the Red CMTPX⁺ population (PC). Cell Tracker dyes have stronger signal than surface markers (conjugated antibodies), but they also bleach faster.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Masanobu Komatsu (mkomats1@jhmi.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Jose L. Herrera (jherre15@jhmi.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate data or code.

Acknowledgments

Fluorescence cell imaging was conducted at the High-Resolution Imaging Core/Shared Resources of Johns Hopkins All Children’s Research Center. Flow cytometry was conducted by Krisztian Csomos at the Flow Cytometry Core of the University of South Florida, St. Petersburg campus. This work was supported by a National Institutes of Health, National Cancer Institute grant (CA125255).

Author contributions

J.L.H. conceptualized and directed the study and designed experiments. J.L.H. performed all experiments and analyzed the data. J.L.H. and M.K. interpreted the data. J.L.H. wrote the manuscript, and J.L.H. and M.K. revised the manuscript. M.K. obtained funding and supervised the study.

Declaration of interests

The authors declare no competing interests.