Protocol for in vitro assessment of human monocyte transendothelial migration using a high-throughput live cell imaging system

Summary

In vitro modeling of the different steps of immune cell recruitment is essential to decipher the role of endothelial cells in this process. Here, we present a protocol for the assessment of human monocyte transendothelial migration using a live cell imaging system. We describe steps for culture of fluorescent monocytic THP-1 cells and chemotaxis plate preparation with HUVEC monolayers. We then detail real-time analysis using the IncuCyte® S3 live-cell imaging system, image analysis, and assessment of transendothelial migration rates.

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

Graphical abstract

Highlights

• •High-throughput in vitro assessment of monocyte transendothelial migration
• •Monitoring monocyte transendothelial migration by real-time imaging
• •Image analysis and quantification of transendothelial migration rates

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

In vitro modeling of the different steps of immune cell recruitment is essential to decipher the role of endothelial cells in this process. Here, we present a protocol for the assessment of human monocyte transendothelial migration using a live-cell imaging system. We describe steps for culture of fluorescent monocytic THP-1 cells and chemotaxis plate preparation with HUVEC monolayers. We then detail real-time analysis using the IncuCyte® S3 live-cell imaging system, image analysis, and assessment of transendothelial migration rates.

Before you begin

The protocol below describes the specific steps for using a THP-1 cell line expressing the far-red fluorescent mKate2-N protein² (named hereafter mKate2-N THP-1 cells), human umbilical vein endothelial cells (HUVECs), IncuCyte® ClearView 96-well chemotaxis cell migration plates (Figure 1), and an IncuCyte® S3 live-cell analysis system placed in a cell incubator.

Note: Although derived from acute monocytic leukemia, the THP-1 monocytic-like cell line is a widespread model of human monocytes because the cells have retained the main characteristics of classical monocytes.³ In addition, an immortalized cell line makes it possible to create a line that stably expresses a fluorescent protein, which is a prerequisite for this method. The method used to obtain the mKate2-N THP-1 cell line and the characteristics of this cell line are described in Ladaigue et al.¹

Note: Cells labeled with commercially available fluorescent dyes could be used instead of THP-1 monocytes expressing a fluorescent protein. This would allow the study of transmigration of primary cells or other cell lines that are difficult to transfect or transduce. However, the stability of the dye, and particularly its ability to be retain in the cell, will have to be checked. In our experience, even a very slight leakage of the dye from THP-1 results in staining of endothelial cells, which leads to a bad segmentation of target objects and can bias the quantification of transmigration rates. We tested two different dyes from Thermo Fisher Scientific (Cell tracker Red CMPTX, ref. C34552, and Cell Tracker Green CMFDA, ref. C7025), which are supposed to be well retained in living cells through several generations. Neither proved satisfactory because their release from the cells resulted in the staining of the HUVECS and consequently in a poor signal-to-noise ratio unusable for image analysis. This part of the protocol deserves to be developed for those who would like to study the endothelial transmigration of cell types other than the THP-1 line, and in particular primary immune cells.

Note: The IncuCyte® ClearView 96-well chemotaxis cell migration plates are composed of three parts: the reservoir plate, the insert plate which contains the insert membranes, and the cover plate (Figure 1A). Each membrane of the insert wells has 96 pores of 8 μm diameter (Figure 1B) to allow the passage of monocytes into the reservoir well below. The wells of the insert plate are seeded with endothelial cells after the membrane is coated with fibronectin. Then, once the endothelial cell monolayer is well established, monocytes are added to initialize the transmigration experiment, as described in Figure 1C. Under these conditions, the growth of HUVECs is very satisfactory and a continuous cell monolayer is formed, as shown in Figure 1D.

Figure 1.

Description of the IncuCyte® ClearView 96-well chemotaxis cell migration plate

(A) The three parts of the chemotaxis cell migration plate. The insert membrane allows transendothelial migration of monocytes through the endothelial cell layer. The reservoir plate allows establishing a concentration gradient of chemoattractant between the two compartments.

(B) Representative images produced by phase contrast microscopy and red fluorescence microscopy of the top and bottom of the permeable membrane of the chemotaxis cell migration plate 24 h after initiation of transendothelial migration in the presence of 100 nM MCP1 in the wells of the reservoir plates. On phase contrast images, THP-1 monocytes appear small, circular, and refractive compared to HUVECs which are larger, more elongated, and less refractive. On red fluorescence scans of the bottom membrane, THP-1 cells on top of the membrane, and therefore not having crossed the HUVEC layer, are visible due to the transparency of the membrane and the HUVECs but appear large and blurred, while monocytes that have crossed, adhering to the membrane, are well focused and are easily identified and segmented by image analysis (see also Figures 2B and 2C). Examples of THP-1 located on the top and the bottom membrane are shown on the red fluorescence scan. The 8-μm diameter pores of the membrane are visible on all three scans. They are marked by green circles on the phase contrast scan of the top membrane and by white circles on the red fluorescence scan of the bottom membrane. Scale bar, 200 μm.

(C) Schematic view of the insert used to perform the transendothelial migration experiment. The membrane of each well has a surface area of 6.68 mm² with 96 pores 8 μm in diameter and is coated with fibronectin. HUVECs form a confluent monolayer. THP-1 are suspension cells which, when seeded in the top part, sediment rapidly and are in contact with HUVECs. The addition of MCP1 in the wells of the reservoir plate establish a concentration gradient of chemoattractant between the two compartments.

(D) Representative phase contrast images of the top of the permeable membrane of the chemotaxis cell migration plate 0, 12 and 24 h after the beginning of the experiment. On these membranes, only HUVECs were seeded, allowing visualization of the state of the endothelial cell monolayer over time. The 8-μm diameter pores of the membrane are visible on all three scans. Scale bar, 200 μm.

Culture of fluorescent monocytic THP-1 cells

Timing: 10 days (for steps 1 to 3)

This section describes a reliable and reproducible method to thaw fluorescent mKate2-N THP-1 cells, and to initiate and maintain their culture for several weeks under satisfactory conditions.

• 1.
  Thaw frozen stocks of mKate2-N THP-1 cells.

  Note: Add 0.05 mM 2-mercaptoethanol to the culture medium to prevent monocyte differentiation into macrophages.

  Note: Thaw mKate2-N THP-1 cells in full RPMI 1640 without the selective geneticin/G418 antibiotic to promote rapid cell culture recovery.

  • a.
    Thaw a tube of mKate2-N THP-1 cells containing 10 × 10⁶ in 1 mL by taking them from a freezer at -150°C (or from liquid nitrogen) and placing the tube immediately in a 37°C water bath for 2–3 min.
  • b.
    Wipe the tube thoroughly with 70% ethanol and pour the contents of the tube into 9 mL of full RPMI 1640 pre-warmed to 37°C.

    • i.
      Centrifuge at 120 × g for 5 min at 20°C–25°C and remove supernatant.
    • ii.
      Resuspend the cells in 1 mL of full RPMI 1640 pre-warmed to 37°C.

      CRITICAL: We use a monocyte cell line that expresses a fluorescent protein. In our experience, as described in the note in the “Before you begin” section, labeling with fluorescent dyes leads to release of the dye, which eventually labels the HUVECs, leading to inaccurate segmentation and quantification of transendothelial migration.

• 2.
  Seed mKate2-N THP-1 cells.

  Note: THP-1 are non-adherent suspension cells that can be grown in fairly large volumes. However, care must be taken not to grow them in too large a volume relative to the size of the flask, as this prevents gas exchange and inhibits cell growth. Typically, use a volume of 15–30 mL in a flask of 75 cm² (T75 flask) in a horizontal position, and a volume of 10 mL in a T75 flask in a vertical position.

  • a.
    Transfer the cell suspension to 39 mL of full RPMI 1640 pre-warmed to 37°C and dispense the culture into two T75 flasks.
  • b.
    Place the T75 flasks horizontally in a cell incubator at 37°C and 5% CO₂ with saturating humidity.
  • c.
    The next day, evaluate the number of living and dead cells using a dye exclusion procedure for viable cell counting.

    Note: We recommend manual cell counting because automatic cell counters are sometimes inaccurate for THP-1. We use cell counting chamber slides, e.g., Malassez counting chamber, and trypan blue as an exclusion dye.

• 3.
  Culture mKate2-N THP-1 cells.

  • a.
    After 3 days of culture, add the selective geneticin/G418 antibiotic to the cell suspension at a concentration of 250 μg/mL.
  • b.
    Culture the cells and maintain the cell culture until the day of the transendothelial migration experiment.

    Note: Dilute the cells to 2 × 10⁵ cells/mL in full RPMI 1640 pre-warmed to 37°C when the culture reaches 8 × 10⁵ cells/mL.

  • c.
    Every 2–3 days, evaluate the number of living and dead cells using a dye exclusion procedure for viable cell counting and check that the number of dead cells is maintained at a maximum of 1%–5% of the total number of cells.

    Note: Restart of cell growth is slow, and significant concentrations of dead cells are expected during the days following thawing, so at least 10 days should be allowed before performing the transendothelial migration experiment. However, once the culture is started, maintain it for several weeks to perform several experiments and avoid this 10-day period. We recommend not to exceed one month of culture, which means a maximum of 8 passages.

Culture of endothelial cells

Timing: 5 days (for steps 4 to 6)

This section describes a reliable and reproducible method to thaw HUVECs, initiate their culture and obtain 80%–100% confluent cell monolayers within five days.

• 4.
  Thaw HUVEC frozen stocks.

  Note: We usually thaw the endothelial cells on Wednesday morning to seed them the following Monday in IncuCyte® ClearView 96-well chemotaxis cell migration plates.

  • a.
    Thaw a tube of HUVECs containing 5 × 10⁵ cells in 0.5 mL by taking them from a freezer at -150°C (or from liquid nitrogen) and placing the tube immediately into a 37°C water bath for 2–3 min.
  • b.
    Wipe the tube thoroughly with 70% ethanol and pour the contents of the tube into 30 mL of EBM™-2 Endothelial Cell Growth Basal Medium-2 containing 5% FBS, supplements and growth factors required for endothelial cells (EGM™-2 MV Microvascular Endothelial Cell Growth Medium-2 SingleQuots™ Kit), hereinafter referred to as full EGM-2 MV, pre-warmed to 37°C.
• 5.
  Seed HUVECs.

  • a.
    Dispense the 30 mL of cell suspension into two T75 flasks.
  • b.
    Place the T75 flasks horizontally in an incubator at 37°C and 5% CO₂ with saturating humidity.
  • c.
    Allow the cells to adhere to the plastic for a minimum of 3–4 h after seeding, then remove the medium and add 15 mL of full EGM-2 MV pre-warmed to 37°C.
• 6.
  Culture HUVEC in an incubator at 37°C and 5% CO₂ with saturating humidity.

  Note: In total, the cells are grown for 5 days from the day of seeding. Under these conditions, the cells reach 80%–100% confluence⁴ and around 80% of the cells are in the G1 phase of the cell cycle.⁵

  • a.
    Two days after seeding, remove the medium and add 15 mL of full EGM-2 MV pre-warmed to 37°C.
  • b.
    Maintain the cell culture for 3 more days until the day of seeding cells in IncuCyte® ClearView 96-Well Chemotaxis Cell Migration Plates.

Seeding HUVECs in IncuCyte® ClearView 96-well chemotaxis cell migration plates

Timing: 1 day (for steps 7 to 9)

This section describes the method of seeding HUVECs in IncuCyte® ClearView 96-well chemotaxis cell migration plates from 80%–100% confluent cell monolayers grown in T75 flasks.

• 7.
  Coat insert membranes with fibronectin.

  Note: We recommend the use of a multi-channel pipette to fill the wells of the reservoir and insert plates.

  • a.
    Prepare a membrane coating solution of 5 μg/mL fibronectin diluted with D-PBS without Ca²⁺ and without Mg²⁺ (D-PBS (−/−)).
  • b.
    Fill the wells of the reservoir plate with 150 μL of the fibronectin solution.
  • c.
    Gently place the insert plate into the reservoir plate.
  • d.
    Fill the inserts with 30 μL of the fibronectin solution.
  • e.
    Incubate for 1 h at 20°C–25°C.
• 8.
  Collect HUVECs.

  Note: During fibronectin coating, HUVECs are collected by trypsinization, counted, and a cell seeding stock of 1 × 10⁵ cells/mL in full EGM-2 MV is prepared.

  Note: The following steps are for 1 T75 flask.

  • a.
    Wash the cell layer using 10 mL of pre-warmed PBS at 37°C, aspirate.
  • b.
    Add 2 mL of pre-warmed trypsin-EDTA solution at 37°C, incubate the flask in the cell incubator at 37°C for 3–5 min.
  • c.
    Add 8 mL of pre-warmed full EGM-2 MV at 37°C, transfer the cell suspension to a 15-mL conical centrifuge tube.
  • d.
    Centrifuge at 200 × g for 5 min and remove supernatant.
  • e.
    Resuspend the cells in 10 mL of full EGM-2 MV pre-warmed to 37°C.
  • f.
    Determine the total number of living cells using a dye exclusion procedure for viable cell counting.

    Note: Any counting method can be used. For this purpose, we use the Bio-Rad TC20 automated cell counter. 20 μL of cells are mixed with an equal volume of 0.4% Trypan blue dye from Bio-Rad, then 10 μL of the mixture is loaded in duplicate onto a slide (Bio-Rad), which is inserted into the cell counter for automatic counting.

  • g.
    Centrifuge at 200 × g for 5 min at 20°C–25°C and remove supernatant.
  • h.
    Resuspend cells at 1 × 10⁵ cells/mL in full EGM-2 MV pre-warmed to 37°C.
• 9.
  Seed HUVECs on top membranes of insert plates.

  Note: Use a multi-channel pipette to fill and aspirate the wells of the reservoir and insert plates.

  Note: Seed cells at 6000 cells (60 μL) per insert well on the membrane surface of 6.68 mm², i.e. at a density of about 9 × 10⁴ cells/cm².

  Note: Prepare at least 4 wells, ideally 6–8 wells, with the same condition/treatment (technical replicates).

  • a.
    Remove the insert plate and place it on a new reservoir plate whose wells contain 200 μL of D-PBS (−/−).
  • b.
    Wash and empty the wells of the insert plate: add 60 μL D-PBS (−/−) to the membrane inserts containing the fibronectin coating solution, aspirate the entire volume.
  • c.
    Immediately seed 60 μL (6000 cells) of the endothelial cell seeding stock at 1 × 10⁵ cells/mL in full EGM-2 MV using a multi-channel pipette into every well of the insert plate (i.e., 6000 cells per insert well).
  • d.
    Allow the cells to settle at 20°C–25°C on a flat surface for 15 min.
  • e.
    Place the IncuCyte® ClearView 96-well chemotaxis cell migration plates containing cells at 37°C and 5% CO₂ with saturating humidity and incubate for 24 h.

    CRITICAL: The IncuCyte® S3 scans the first left top well (in position A1) with a large Z-scan, which will be used as the basis for the next scan to make a smaller Z-scan. All other scans will then be very fine. This first well must be filled with cells, otherwise the following Z-scans will be logically badly calibrated.

    CRITICAL: When filling and especially emptying the insert wells with the multichannel pipette, care must be taken not to damage membranes by touching them too roughly. When aspirating, we recommend leaving a small volume of solution in the insert well to avoid touching the membrane.

    CRITICAL: Repeat the experiment and become proficient at it until you obtain endothelial cell sheets of consistent quality every time.

Treatment of the HUVEC monolayers (optional)

Timing: 1–2 days (for step 10)

This section shows additional steps to treat HUVEC monolayers grown in IncuCyte® ClearView 96-well chemotaxis cell migration plates to study effects of interest. It presents an example of such a treatment for studying the consequences of ionizing radiation on transendothelial migration.

• 10.
  Treatment of the HUVEC monolayers.

  Note: At this stage of the protocol, HUVEC monolayers can be exposed to various treatments, such as cytokines, chemokines, antibodies, chemicals or electromagnetic fields, or gene expression modifications by transfection or transduction in order to investigate mechanisms of transendothelial migration. Here, we present an example of treatment by exposure to ionizing radiation, which results in an increased rate of transendothelial migration.¹

  Note: The time between the start of treatment and the initiation of transendothelial migration should not exceed 2 days, as cells seeded at near-confluence density may be adversely affected. We stress here that each treatment must be tested beforehand so that the experimental conditions are optimal.

  • a.
    Aspirate the cell culture medium and add 60 μL of full EGM-2 MV pre-warmed to 37°C.
  • b.
    Irradiate the cells (for a complete and detailed protocol, see Ladaigue et al.¹).
  • c.
    Place the plates containing cells at 37°C and 5% CO₂ with saturating humidity and incubate for 24–48 h.

    CRITICAL: When removing culture medium from insert plates, we recommend leaving a small volume of medium to avoid damaging the HUVEC monolayers. Change the culture medium two columns by two columns so that the cells are not in contact with air for too long, which could result in damage or death.

    CRITICAL: If treatment results in endothelial cell damage, an increase in transmigration could be explained by loss of monolayer integrity and not by changes in endothelial cell phenotype. The experiment should therefore include a check on the integrity of the monolayer. For example, the integrity of the HUVEC monolayer can be assessed by a permeability study using Dextran-FITC, as we did in our study on the effect of ionizing radiation.¹

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s Phosphate Buffered Saline	Thermo Fisher Scientific	Cat#: 14190-094
EBM™-2, Endothelial Cell Growth Basal Medium-2	Lonza	Cat#: CC-3156
EGM™-2, MV Microvascular Endothelial Cell Growth Medium-2 SingleQuots™ Kit	Lonza	Cat#: CC-4147
RPMI 1640 Medium (ATCC Modification)	Thermo Fisher Scientific	Cat#: A10491-01
Gibco™ Fetal Bovine Serum, qualified, E.U.-approved, South America origin	Thermo Fisher Scientific	Cat#: 10270106
2-Mercaptoethanol	Thermo Fisher Scientific	Cat#: 21985023
Trypsin-EDTA	Thermo Fisher Scientific	Cat#: 25300054
Trypan blue dye	Bio-Rad	Cat#: 145-0013
Cell counting slides	Bio-Rad	Cat#: 145-0011
Penicillin-Streptomycin	Thermo Fisher Scientific	Cat#: 15140122
Human monocyte chemoattractant protein 1 (MCP1)/ monocyte chemotactic and activating factor (MCAF)	Sigma-Aldrich	Cat#: SRP3109-20UG
Fibronectin bovine plasma	Sigma-Aldrich	Cat#: F1141-1MG
IncuCyte® ClearView 96-well chemotaxis plate	Sartorius	Cat#: 4648
Geneticin™ Selective Antibiotic (G418 Sulfate)	Thermo Fisher Scientific	Cat#: 10131035
pmKate2-N	Euromedex	Cat#: FP182
Experimental models: cell lines
HUVECs – Human Umbilical Vein Endothelial Cells	Lonza	Cat#: C2519A
mKate2-N THP-1 monocyte cells	Ladaigue et al.1	https://doi.org/10.1016/j.isci.2022.105482
Software and algorithms
GraphPad Prism version 8.1.1	GraphPad Software	www.graphpad.com
IncuCyte® software v2022RevB	Sartorius	www.sartorius.com
IncuCyte® Chemotaxis Analysis Software Module	Sartorius	Cat#: 9600-0015
ImageJ version 1.52a software	National Institutes of Health	http://imagej.nih.gov/ij
Other
Ultra-low temperature freezer (−150°C) (alternative: liquid nitrogen tank)	PHCBI	Cat#: MDF-C2156VAN
T75 culture flasks	Falcon	Cat#: 353136
1.8 mL cryogenic microtubes	Thermo Scientific	Cat#: 363401
15- and 50-mL conical centrifuge tubes	Falcon	Cat#: 352096 Cat#: 352070
1.5 mL microcentrifuge tubes	Eppendorf	Cat#: 0030123328
5-, 10- and 25-mL pipettes	Falcon	Cat#: 357543 Cat#: 357551 Cat#: 357525
0.1–10, 2–200, 50–1000 μL micropipette tips	Eppendorf	Cat#: 0030000811 Cat#: 0030000889 Cat#: 0030000927
0.1–2, 2–20, 20–200 and 100–1000 μL micropipettes	Rainin	Cat#: 17008648 Cat#: 17008650 Cat#: 17008652 Cat#: 17008653
300 μL multi-channel micropipette	Eppendorf	Cat#: 3125000060
Water bath	Julabo	Cat#: 9550112
Centrifuge for microtubes	Eppendorf	Cat#: 5430R
Centrifuge for 15/50 mL conical tubes	Eppendorf	Cat#: 5810R
Plate centrifuge (optional)	Eppendorf	Cat#: 5804
Biological safety cabinet (BSC)	Thermo Scientific	Cat#: 51025411
Malassez counting chamber	VWR	Cat#: HECH40453702
Bio-Rad TC20 automated cell counter (optional)	Bio-Rad	Cat#: 1450102
Cell counting slides, dual chamber for TC20 cell counter (optional)	Bio-Rad	Cat#: 1450011
Cell culture incubator	PHCBI	Cat#: MCO-170AICUVH
Cell culture incubator for IncuCyte® S3 live-cell analysis instrument (Heracell 240i)	Thermo Fisher Scientific	Cat#: 16426639
Phase-contrast microscope	Zeiss	Cat#: Axiovert35
IncuCyte® ClearView 96-Well Chemotaxis Cell Migration Plates	Sartorius	Cat#: 4582
IncuCyte® ClearView 96-Well Chemotaxis Cell Migration Reservoir Plates (optional)	Sartorius	Cat#: 4600
Incucyte vessel trays	Sartorius	Cat#: 5025-0116
IncuCyte® S3 live-cell analysis instrument	Sartorius	Cat#: 4647

Materials and equipment

Cell culture media

Full RPMI 1640

Reagent	Final concentration	Amount
RPMI 1640 Medium (ATCC Modification)	N/A	500 mL
Heat inactivated Fetal Bovine Serum	10%	57 mL
Penicillin-Streptomycin	2%	11.4 mL
2-mercaptoethanol	0.05 mM	0.57 mL
Total	N/A	569 mL

Heat-inactivate fetal bovine serum by heating serum in a water bath at 56°C for 20 min and store at −20°C in 28.5 mL aliquots. Full RPMI 1640 can be stored for up to 3 months at 4°C.

Full EGM-2 MV

Reagent	Final concentration	Amount
EBM™-2 Endothelial Cell Growth Basal Medium-2	N/A	500 mL
Fetal Bovine Serum	5%	25 mL
Hydrocortisone	Not available	0.2 mL
Human Fibroblast Growth Factor-Beta (hFGF-β)	Not available	2 mL
Vascular Endothelial Growth Factor (VEGF)	Not available	0.5 mL
R3-Insulin-like Growth Factor-1 (R3-IGF-1)	Not available	0.5 mL
Ascorbic Acid	Not available	0.5 mL
Human
Epidermal Growth Factor (hEGF)	Not available	0.5 mL
Gentamicin/Amphotericin-B (GA)	Not available	0.5 mL
Total	N/A	529.7 mL

The concentrations of supplements and growth factors are not communicated by Lonza. Full EGM-2 MV can be stored for up to 3 months at 4°C.

Step-by-step method details

Monitoring monocyte transendothelial migration by real-time imaging

Timing: 1–3 days (for steps 1 to 3)

This section accomplishes the initialization and the monitoring of transendothelial migration by real-time microscopic imaging.

We describe the initialization of transendothelial migration and the monitoring of the experiment by real-time microscopic imaging. This entails filling the inserts with fluorescent THP-1 in full EGM-2 MV medium, filling reservoir wells with full EGM-2 MV containing the monocyte chemoattractant protein 1 (MCP1)/monocyte chemotactic and activating factor (MCAF) (hereafter referred to as MCP1) and launching the scan acquisition program.

• 1.
  Preparation of mKate2-N THP-1 cells.

  Note: Seed mKate2-N THP-1 cells in the IncuCyte® ClearView 96-well chemotaxis cell migration plates at 5000 cells/well in 60 μL of HUVEC culture medium (full EGM-2 MV), i.e. at a concentration of 8.33 × 10⁵ cells/mL.

  CRITICAL: Prepare twice the required number of fluorescent mKate2-N THP-1 cells to anticipate losses due to trapping of cells by the plastic wall of the tubes during preparation and centrifugation.

  • a.
    Determine the concentration of mKate2-N THP-1 cells in the culture by a dye exclusion procedure for viable cell counting.
  • b.
    Centrifuge twice the required number of cells at 120 × g for 5 min at 20°C–25°C, then remove supernatant.
  • c.
    Resuspend the cells in half the theoretical volume of full EGM-2 MV pre-warmed to 37°C to obtain a final concentration of 8.33 × 10⁵ cells/mL.
  • d.
    Determine the total number of mKate2-N THP-1 cells by a dye exclusion procedure for viable cell counting.
  • e.
    Add full EGM-2 MV pre-warmed to 37°C to reach a final concentration of 8.33 × 10⁵ cells/mL.
  • f.
    Store at 37°C until seeding in insert plates.
• 2.
  Seeding mKate2-N THP-1 cells in IncuCyte® ClearView 96-well chemotaxis cell migration plates.

  • a.
    Rinse HUVEC monolayers with 60 μL of EGM-2 MV pre-warmed to 37°C and empty the wells.
  • b.
    Add 60 μL (5000 cells) of mKate2-N THP-1 cells per well.
  • c.
    Centrifuge plates at 50 × g for 3 min at 20°C–25°C to rapidly bring monocytes to the surface of the endothelial cell monolayer.

    Alternatives: This step requires a plate centrifuge. If centrifugation is not possible, allow mKate2-N THP-1 cells to settle on the endothelial monolayer at 20°C–25°C for 45–60 min. In this case, and for each type of cell, it will be necessary to determine the minimum time to allow for sedimentation to standardize the start time of the experiment.

  • d.
    Fill the wells of a new reservoir plate with 200 μL of full EGM-2 MV pre-warmed to 37°C and containing 100 nM of the chemoattractant MCP1.
  • e.
    Transfer the insert plate containing the cells into the new pre-filled reservoir plate containing full EGM-2 MV ± MCP1.

    CRITICAL: When removing culture medium from insert plates, we recommend leaving a small volume of medium in the insert well to avoid touching and damaging the HUVEC monolayers.

    CRITICAL: Change the medium two columns by two columns so that the cells are not in contact with air for too long, which could result in damage or death. Use the same procedure to fill the wells with mKate2-N THP-1 cells.

• 3.
  Launching real-time acquisitions with the IncuCyte® S3 live-cell analysis system and analysis of the scans.

  • a.
    Place the IncuCyte® ClearView 96-well chemotaxis cell migration plates on the vessel tray placed in the IncuCyte® live-cell analysis instrument (which itself is placed in a Heracell 240i cell incubator) and allow the plate to warm to 37°C for about 10 min.
  • b.
    If necessary, wipe away any condensation remaining on the outside of the cover plate.
  • c.
    In the chemotaxis module of the IncuCyte® software, schedule 24-h scans every 30 min for one plate for a total time of 3 days.

Note: The scan time for one plate is approximately 26 min. Adapt the frequency of the scans to the number of plates, i.e. every hour for 2 plates or every two hours for 4 plates. In our experiments, scans of the top and bottom sides of the permeable membrane are recorded in transmitted light and red fluorescence (800 ms exposure) using a ×10 objective lens.

Note: The chemotaxis module scans each well in its entirety in a single image. There is no other possibility.

Note: The choice of exposure time depends on the fluorescence intensity of the cells and should be adjusted beforehand. However, once adjusted for a cell line, use this value for each new experiment.

CRITICAL: Long scans cause a significant increase in temperature inside the IncuCyte® S3 device. The Status window provides options for viewing the temperature. To maintain a constant temperature of 37°C within the IncuCyte® S3 instrument, lower the temperature of the cell incubator. Trials are necessary to find the right temperature. We usually lower the temperature of the cell incubator to 35.8°C for scanning 2 plates every hour.

Image analysis and assessment of transendothelial migration rates

Timing: 1–2 h (for steps 4 to 6)

This section accomplishes the analysis of the transendothelial migration data acquired with the IncuCyte® S3 instrument.

We describe here the steps for analyzing transendothelial migration data acquired over time with the IncuCyte® S3 instrument.

Note: The IncuCyte® device performs phase and fluorescence scans focused on the top surface of membranes (on which endothelial cells and THP-1 fluorescent monocytes are located) and fluorescence scans focused on the bottom of the membrane (on which monocytes that have passed through the endothelial cell monolayer and the membrane pores under the influence of the chemotactic gradient remain attached to the membrane, at least temporarily, and can be visualized).

In our experiment, detection of fluorescent objects is achieved by setting the appropriate segmentation parameters from the scans of the bottom of the membranes. Fluorescent THP-1 cells on top membranes are not studied. Quantification of top fluorescent THP-1 is indeed too inaccurate due to the density of THP-1 in the upper compartment (5000 cells per 6.68 mm²), which alters segmentation, and due to cell multiplication over time, which continuously changes the number of cells. Furthermore, the number of fluorescent objects detected over time on the bottom of the membrane is quite small compared to the number of cells seeded (about 10%), making it easier and more accurate to track the appearance of cells that have migrated through the endothelial cell monolayer rather than those that persist.

Note: The chosen parameters allow segmenting the fluorescent objects that have undergone transendothelial migration, even if those that remained on the top of the membrane are visible because of the transparency of the membrane and of the endothelial cells (Figure 2). The focus is placed on these objects which appear smaller and sharper than those on the top of the membrane, as the latter are blurred and broader in appearance, making it possible to assign segmentation parameters for their exclusive detection (Figures 2B and 2C).

CRITICAL: It is important to correctly define the parameters for segmenting the fluorescent objects which are applied to each scan. Within our laboratory, the transendothelial migration experiments are very reproducible, and the parameters have almost never been changed, allowing the saved analysis parameters to be reused to perform the analysis automatically as the experiment progresses and to save time. We describe the segmentation parameters we commonly use in this section, in part 4b. However, it is necessary to visually verify that the fluorescent objects are correctly detected from one experiment to another with these analysis parameters. Otherwise, refine these parameters to launch a new analysis on the same data.

Note: The “Quantification and statistical analysis” section describes the meaning of the segmentation parameters and how they can be set.

• 4.
  Scan analysis.

  Note: Once the scan parameters are set, they can be saved and reused, and the analysis can be performed automatically after each scan during the acquisition period. For this purpose, at the time of launch, load the parameters of the pre-saved analysis in the Analysis Setup window.

  • a.
    Use the IncuCyte® software (Chemotaxis Migration Top/Bot analysis) to perform the scan analysis.
  • b.
    Use the following analysis settings as a basis for determining segmentation parameters: for the phase, seed threshold of 50, grow threshold of 100, no clean-up, and no area and eccentricity filters; for the red fluorescence, segmentation with a seed threshold (RCU) of 10^-5, a grow threshold of 50, an edge sensitivity of 0, no clean-up filter, area filter of minimum 20 mm² and maximum 600 mm², eccentricity filter of maximum 0.78, mean intensity filter of 0.1, and no integrated intensity filter.

    CRITICAL: The segmentation parameters are to be established in each laboratory because they are probably dependent on the cells used and the laboratory in which the experiment is performed. We show here the parameters that we commonly use. A detailed method for determining the segmentation and filtering parameters is described below in the “Quantification and Statistical Analysis” section.

• 5.
  Data analysis and visualization.

  • a.
    Export the data from the IncuCyte® software.

    • i.
      In analysis results, open the graph data.
    • ii.
      Select [Bottom] metrics, wells, and scans to be considered for export.
    • iii.
      In the selecting grouping menu, check “None” and click on “Export data.”

      Note: Exported the data to a text file and open it with a spreadsheet program such as Microsoft Excel.

  • b.
    Analyze the data in statistical software.

    • i.
      Open the export text file with a spreadsheet program such as Microsoft Excel, and organize the data as required for the statistical analysis.
    • ii.
      Import the data into statistical analysis software such as GraphPad Prism.
    • iii.
      Analyze and represent the data in visualization charts.
• 6.
  Visualization of transendothelial migration of monocytes in video form.

  Note: The IncuCyte® software enables exporting images and movies from the same well. This can be done from unanalyzed and analyzed scans, which in the latter case allows displaying the masks of the objects and thus visualizing the segmented objects at each time point.

  • a.
    After opening an experiment or an analyzed experiment, click on the Export Images and Movies icon.
  • b.
    In the Export Type window, choose “As Displayed.”
  • c.
    In the Wells Selection window, select one or more wells and choose the Image Channel(s) and the Channel Mask(s) to display.
  • d.
    In the Sequence Type and Scans window, select “Single Movie of Images as Displayed” and select the scans to include in the movie.
  • e.
    In the Export Design window, adjust cropping and scaling, customize the movie, and choose the number of frames per second.
  • f.
    Export movies in MPEG-4 or AVI video file format.
  • g.
    To edit the videos, import the movies in AVI file format into Image J software. Examples of movie editing are displayed in Methods videos S1 and S2.

Figure 2.

The segmentation parameters allow rigorous selection of fluorescent THP-1 cells that have crossed the endothelial cell layer and the membrane

The figure shows red fluorescence scans of the same field of the top and bottom of the permeable membrane of the chemotaxis cell migration plate and the segmentation results for the fluorescent objects after analysis by the IncuCyte® software (24 h after initiation of transendothelial migration in the presence of 100 nM MCP1 in the wells of the reservoir plates).

(A) Control panel for viewing scans and segmented objects. The panel allows choosing the time point, the scan (phase or red, top or bottom), and the masks (top red, bottom red, pore) to be viewed in the window on the right of the panel.

(B and C) Red fluorescence channel images of the top and the bottom of the membrane without (B) and with (C) segmentation masks. In the bottom channel image, THP-1 on top of the membrane, and therefore not having crossed the HUVEC layer, appear large and blurred, while monocytes that have crossed, adhering to the membrane, are well focused and detected by the IncuCyte® S3 segmentation module (circled in yellow). Similarly, in the top channel image, THP-1 cells on top of the membrane are efficiently segmented by the software (circled in purple). Examples of THP-1 located on the top and the bottom membrane are shown on the red fluorescence scan. Scale bar, 200 μm.

Expected outcomes

In our experiments, the number of THP-1 detected under the membrane increases continuously and linearly over time for the first 20–24 h, then the curve bends and this number reaches a plateau. A maximum number is reached in 24–48 h, is dependent on the concentration of the chemoattractant MCP1 in the reservoir wells, and reaches about 200–300 events in normal conditions for a concentration of 100 nM MCP1. This maximum number can double (about 600 events) when endothelial cells are previously exposed to ionizing radiation at a dose of 20 Gy. See Figure 3 for example data from experiments with increasing concentrations of chemoattractant and increasing doses of ionizing radiation. See Methods videos S1 and S2 for real-time visualization of THP1 passage through membrane pores and THP-1 detection by IncuCyte® software in the absence or presence of MCP1 chemoattractant, and after exposure or no exposure to ionizing radiation.

Figure 3.

Transendothelial migration of THP-1 monocytes as a function of MCP concentration and irradiation dose

(A) Number of mKate2-N THP-1 cells detected under the membrane by the IncuCyte® S3 module from 0 to 72 h after initiation of transendothelial migration and at different concentrations of MCP1 in the reservoir plate (from Ladaigue et al.¹). HUVECs were seeded on the wells of an IncuCyte® ClearView 96-well chemotaxis cell migration plate. Seventy-two hours after the seeding of the HUVECs, mKate2-N THP-1 cells were added to the well, and culture medium containing 0, 12.5, 25, 50, 100, or 200 nM MCP1 was added to the wells of the plate reservoir. The plates were placed in the IncuCyte® S3 live cell analysis system in a cell culture incubator to monitor the transendothelial migration of monocytes every hour for 3 days. The images from the experiment can be exported and edited into a movie using IncuCyte® software and Image J (see Methods video S1).Data are represented as mean +/− SEM derived from n = 8–16 technical replicates.

(B) Number of mKate2-N THP-1 cells detected under the membrane by the IncuCyte® S3 module from 0 to 72 h after initiation of transendothelial migration and after irradiation of HUVECs at different doses, in the absence (-MCP1) or in the presence (+MCP1) of 100 nM MCP1 in the wells of the reservoir plates (from Ladaigue et al.¹). HUVECs were seeded on the wells of an IncuCyte® ClearView 96-well chemotaxis cell migration plate. Twenty-four hours after the seeding of HUVECs, the plates were irradiated at 0, 2, 5, 10, or 20 Gy. Forty-eight hours after irradiation, mKate2-N THP-1 cells were added to the wells, and culture medium containing 100 nM MCP1 was added to the wells of the plate reservoir. Plates were placed in the IncuCyte® S3 live cell analysis system in a cell culture incubator to monitor the transendothelial migration of monocytes every 1.5 h for 3 days. The images from the experiment can be exported and edited into a movie using IncuCyte® software and Image J (see Methods video S2).Data are represented as mean +/− SEM derived from n = 8–16 technical replicates.

(C) The graphs present the quantification of transendothelial migration of THP-1 mKate2 cells as a function of MCP1 concentration 24, 48, and 72 h after initiation of the transmigration (from Ladaigue et al.¹). Data are represented as mean +/− SEM derived from n = 8–16 technical replicates. One-way ANOVA test with Tukey correction; ns, not significant, ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001.

Quantification and statistical analysis

To quantify transendothelial migration rates, the definition of the analysis parameters must be carefully adjusted. This section describes the different steps to achieve this correctly. The segmentation parameters must be set in each laboratory and for each type of experiment as they are highly dependent on the fluorescent cell line used and other conditions. These parameters can be recorded for reuse in subsequent experiments; once set up, the experiment is highly reproducible. Perfect detection of THP-1 should not be sought, as it is impossible to achieve in real conditions with the tools and equipment available. One reason for this is that the fluorescence intensities vary from cell to cell in the culture of the fluorescent THP-1 line, ranging from high-intensity to almost imperceptible fluorescence (Figure 4), although this line is cloned from a cell sorted by FACS based on its ability to emit high fluorescence intensity.¹

Figure 4.

The fluorescence intensity of mKate2-N THP-1 cells varies from cell to cell

This figure shows a representative image of mKate2-N THP-1 cells grown in 6-well plates obtained by scanning in the red fluorescence channel using an exposure time of 800 ms with the IncuCyte® imaging system. Although a clone was obtained from a transduced cell (see Ladaigue et al.¹), variations in fluorescence intensity from one cell to another are observed (left panel). However, the segmentation parameters of the Incucyte® S3 allow the detection of all cells present in the microscopy field, as shown in the right panel which presents the same image as in the left panel after segmentation (segmented mKate2-N THP-1 cells are circled in blue). Scale bar, 200 μm.

To set segmentation parameters, it is first necessary to determine the parameters that minimize the detection of false positives. Then, if possible, the parameters that allow for maximum detection of THP-1 are determined. It is not very important that not all THP-1 are detected, as we are looking for relative comparisons. However, it is obviously important that the detection is homogeneous between the wells and the different plates.

Quantification of the transendothelial migration rate is performed after segmentation and filtration of fluorescent objects. Below, we describe the basic principles we use for this step, based on Sartorius’ recommendations and our own experience. To establish a new analysis definition, we recommend the following step-by-step procedure.

• 1.Open an experiment (vessel), launch the analysis, choose “Create a new analysis definition” and “Chemotaxis migration (Top/Bot)” as analysis type, and select at least the Red channel.
• 2.
  At the step of image set selection, we recommend choosing only one image at one time point in which monocytes that have transmigrated are seen but not in too large numbers. This image should be representative of the overall images after several images are viewed and at several analysis time points.

  Note: We usually choose a scan performed around 24 h after transmigration initialization. At this time, the plateau has not yet been reached and THP-1 cells have not yet begun to multiply significantly in the upper compartment, an effect that could render the assay definition parameters inaccurate due to excessive fluorescence.

• 3.Adjusted the settings n the “Red bottom” image channel chosen in the Layers menu.
• 4.
  Segmentation.

  Note: As described for the interface of the IncuCyte® software, a Seed-and-Grow technique is used to mask the cells from the background (Figure 5A). This technique uses two thresholds, the Seed Threshold and the Grow Threshold.

  • a.
    Select the Seed Threshold first.
  • b.
    Start with a Red Calibrated Unit (RCU) of the objects at 0 (default).
  • c.
    Launch a preview.
  • d.
    Use the mouse pointer to open the tooltip to evaluate the RCU of the non-segmented red objects or the wrongly segmented objects (Figure 5A, image on top right).
  • e.
    Change the Seed Threshold value to reflect the estimation by the mouse pointer and launch a preview.
  • f.
    Repeat steps d–e until as many objects as possible are correctly detected.
  • g.
    Set the Grow Threshold value until a satisfactory segmentation is obtained.
  • h.
    Finally, adjust the Edge Sensitivity value to split different objects that would otherwise be one object (Figure 5B).
• 5.Clean up.

  IncuCyte® software offers the ability to fill the holes that may have been left in the objects (Holl Fill (μm²)), as well as to adjust the size of the objects (Adjust Size (pixels)) to the actual fluorescence area visualized (Figure 5C). At this stage of the analysis definition, these parameters do not change the number of detected objects but allow a better visualization and a more accurate quantification of the fluorescence intensity of the segmented objects. For the quantification of transmigration rates, based only on the number of objects, these parameters have no impact.

• 6.Filter.

  Once segmented, objects can be selected using 4 filters based on area, eccentricity (i.e., shape), mean intensity and integrated intensity. For each of these filters, minimum, maximum, or both values can be defined (Figure 5D). The parameter values of each object can be visualized by means of the mouse pointer to open the tooltip on the setting image. The use of filters obviously changes the number of detected objects.

• 7.
  Select scan times and wells to consider, and save and apply analysis definition to create the analysis.

  Note: We found high reproducibility of object size, fluorescence, and signal-to-noise ratio across experiments, allowing the same analysis definition parameters to be used for the different experiments. As already mentioned, however, it is necessary to verify that the segmentation is satisfactory after the analysis is complete.

• 8.Statistical analysis.
  The IncuCyte® software includes a graph builder and a graph viewer that allows visualizing the curves of the number of detected objects for the different conditions versus time. This allows generating two types of graphs, one to visualize the values of each well as a function of time (Microplate Graph) and the other to visualize the means and standard deviations in each condition as a function of time (Graph) (Figure 6). The graphs of mean values versus time are useful to track the experiment in real time, provided that the analysis is performed at the end of each scan (reuse of a saved analysis definition). They are also useful to quickly visualize the results after all scans have been analyzed. The Microplate Graph is used to visualize the data from each well and to identify data from the wells (i.e., technical replicates) that should be excluded from the analysis due to technical problems. Statistical tests and graphical representation of data for publications are done in statistical analysis software such as GraphPad Prism after exporting data from IncuCyte® software.

  • a.
    Number of technical replicates.
    The curves are highly similar from well to well (see, for example, Figure 6, Microplate Graph); we nevertheless recommend n = 6–8 technical replicates per condition of interest. This large number of replicates is permitted using 96-well plates. If wells need to be excluded from the analysis, this allows sufficient data to be retained for statistical analysis. For basic controls whose data are not involved in the quantitative analysis, such as wells without THP-1 or wells without chemoattractant, n = 4 technical replicates are sufficient.
  • b.
    Criteria for data inclusion/exclusion.
    The Microplate Graph Viewer can be used to identify wells where a technical problem has occurred and interfered with the normal course of transendothelial migration. These problems occur at a very low frequency. They may be due to design flaws in the membrane (very rare), extremely rarely impurities in the well (introduced by the experimenter or the culture medium), defects in the endothelial cell monolayer (e.g., due to growth problems or damage caused by the experimenter during handling), experimenter errors (such as forgetting to add the THP-1 cells or the treatment product), or problems during scan acquisition (troubleshooting). Most of the problems mentioned here can be avoided by good laboratory practices but there are other issues that cannot be avoided, such as defects in the membranes or errors made by the device during scans. Figure 7 gives some examples of technical problems and the visualization of the data that help to identify them (see also the troubleshootingsection). Scans that are identified as incorrect due to proven technical problems are excluded from the analysis.
  • c.
    Graphical representation of data and statistical analysis.
    Data exported in .csv format are opened in a Microsoft Excel spreadsheet to organize them, then pasted into statistical analysis software such as GraphPad Prism to build graphs and perform statistical tests.
    The most useful graphs present curves of the average number of THP-1 objects as a function of time (Figures 3A and 3B), as well as individual values of the number of THP-1 objects at given times after transendothelial migration initialization in scatter plots with bar (Figure 3C).
    To find out whether the means of the conditions differ statistically, classical parametric (ANOVA) or non-parametric (Kruskal-Wallis test) statistical tests with correction for multiple comparisons are performed at different times, e.g., at 24, 48, and 72 h.

Figure 5.

Establishing a new analysis definition: segmentation, clean-up, and filtering

(A) The analysis definition interface (left side of the left image) is first used to determine the segmentation parameters using a Seed-and-Grow technique (see on the figure the description given by the software through the tooltip system). The information indicated by the tooltip when the mouse pointer hovers over a fluorescent THP-1 cell (image on top right) is used to adjust the Seed Threshold level (in RCU units). By making several adjustments to the Seed Threshold, a satisfactory result can be achieved.

(B) The segmentation is refined by determining parameters that allow separating the objects that need to be separated with the Edge Split tool. An example of the effectiveness of this tool is shown in the two images on the right.

(C) Cleanup parameters are used to improve the segmentation masks. An example of the effect of the Adjust Size (Pixels) parameter is shown in the two images on the right.

(D) Once segmented, the objects can be filtered using the four parameters proposed by the software interface, namely the Area, the Eccentricity, the Mean Intensity, and the Integrated Intensity.

Figure 6.

Create graphs with the IncuCyte® software

The IncuCyte® software includes a graph builder and a graph viewer that allows visualizing the curves of the number of detected objects for the different conditions versus time. This allows generating two types of graphs, one to visualize the values of each well as a function of time (Microplate Graph), and the other to visualize the means and standard deviations in each condition as a function of time (Graph). In this experiment, there are 12 experimental conditions, with 8 technical replicates per condition arranged in the 8 wells of columns 1 to 12. The graphs of mean values versus time are useful to track the experiment in real time or to quickly visualize the results after all scans have been analyzed. The Microplate Graph is used to visualize the data from each well and to identify data from the wells (i.e., technical replicates) that should be excluded from the analysis due to technical problems. In the graph, data are represented as mean +/− SD derived from n = 8 technical replicates.

Figure 7.

Main technical problems identified by the analysis of scans and data

This figure shows the most frequently encountered problems. The two main causes of technical problems are human causes (e.g., cell seeding errors, damage to the membrane or endothelial cell layer) or IncuCyte® scanning errors. In general, these problems are rare, and most of the time it is sufficient to exclude data from one or a few wells at some or all time points.

(A) In this experiment, there are 8 technical replicates per condition arranged in the 8 wells of each column. The Microplate Graph shows an atypical curve of THP-1 count versus time in well B1 (circled in red) compared to other replicates. In this well, the scan shows that the endothelial cell monolayer is poorly formed (non-confluent cells) due to an unidentified problem, such as a seeding error or a defective membrane. The best solution is to exclude the data from this well.

(B) In this experiment, there are 6 technical replicates per condition arranged in the rows. The Microplate Graph shows heterogeneous curves of THP-1 count versus time in wells A7-12 and B7-12 (circled in red). In two of these wells, scans show that there are no THP-1 cells. More generally, it appears that there was a problem with the distribution of THP-1 cells at the time of seeding. Here, the best solution is to repeat the experiment.

(C) In this experiment, there are 4 technical replicates per condition arranged in the columns. The Microplate Graph shows problems in the THP-1 number versus time profiles in wells A4 and A5 (circled in red). The scans reveal a problem with the scan acquisition for these wells. Excluding data from these wells improves the plots of mean THP-1 counts versus time, as shown in the bottom graphs. Data are represented as mean +/− SD derived from n = 4 technical replicates.

(D) An example of a scan acquisition problem for a well (circled in red), for some unknown reason. Here it is sufficient to exclude the data from this replicate.

(E) Sometimes only one scan fails. In this experiment, there are 8 technical replicates per condition arranged in the 8 wells of the columns. The microplate graph shows atypical THP-1 counts at specific time points in wells B9 and C1 (circled in red). In these wells, the scans display an error in the acquisition of a part of the membrane area (framed in red). The best solution is to exclude the data from these time points.

Limitations

Since this is an in vitro assay, the limitations are related to culture conditions, stimulation and observation times, chemoattractant type and concentration, and cell types that may affect transmigration efficiency. The cell culture is far from the physiological context in vivo, especially since it lacks the other tissue cell types, the extracellular matrix, blood flow and its constituents, and the subendothelial basement membrane. After an exploratory phase with this high-throughput system, it will be important to conduct studies to identify candidate factors that influence transendothelial migration using a system with cells under flow. Furthermore, the current setup allows measurement of changes in two phenomena together, chemokinesis and chemotaxis, without knowing their respective contributions, as allowed for example by the protocol published by Schoppmeyer and van Buul.⁶

Troubleshooting

The only problems we identified were at the transendothelial migration step, for at most a few wells, either at a single time point or at multiple time points. Microplate graphs and scan visualization help reveal these problems. The only solution is the objective and justified exclusion of data from defective scans if the problem is clearly identified. The large capacity of the IncuCyte® chemotaxis cell migration plate (96 wells) allows for many replicates per condition, which is useful to anticipate the exclusion of some wells in case of technical problems. For the conditions of interest, other than standard control conditions such as wells without MCP1, without THP-1, or without HUVECs , we therefore recommend n = 6–8 technical replicates per condition.

Problem 1

Absent, non-confluent or damaged endothelial cell monolayer or damaged membrane revealed by too many THP-1 cell migrations in one well compared to other replicates of the same condition (Figure 7A).

Potential solution

• •Repeat the experiment and become proficient at it until you obtain endothelial cell sheets of consistent quality every time.
• •Take care not to damage the endothelial cell monolayer or the membrane when changing medium or adding THP-1 cells.

Problem 2

Absence of THP-1 cells or wrong concentration compared to the other wells, decrease in the fluorescence of the clone cells in all wells, or poor definition of analysis parameters (segmentation and filtration) revealed by no or too few THP-1 cell migrations in one or several wells compared to other replicates of the same condition, or in all wells (Figure 7B).

Potential solution

• •Make sure to add the correct number of THP-1 cells to each well.
• •Make sure to add MCP1 to each bottom well.
• •In case of a segmentation problem, redefine the parameters and launch a new analysis on the same data (not lost). To redefine the parameters, refer to the “quantification and statistical analysis” section.
• •In case of an inherent problem with the clone fluorescence, repeat the experiment after thawing a new ampoule of red fluorescent THP-1.

Problem 3

Problem of scan acquisition revealed by an aberrant curve of the number of THP-1 migrations over time, with very heterogeneous values, or by highly aberrant values at specific times (Figures 7C–7E).

Potential solution

• •Clearly identify the problem by examining the scans. The z-positions where the Incucyte focuses can be exported for examination to identify focusing problems. This method can exclude data points that have a very different z-position.
• •Exclude from the analysis the time points where the scans are incorrect, or all the scans of a well if all time points are concerned.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Olivier Guipaud (olivier.guipaud@irsn.fr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request.

This study did not generate datasets.