A Laminar Flow-Based Assay to Study the Interactions Between Leukocytes and Endothelial Cells

Published: January 31, 2024

Abstract

Source: Vajen, T., et al. Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets. J. Vis. Exp. (2018).

This video demonstrates a laminar flow-based assay to characterize the interaction of leukocytes and cellular partners such as endothelial cells. This method provides dynamic insights into adhesion, deadhesion, and transmigration of leukocytes when perfused over an intact and confluent layer of vascular cells.

Protocol

1. Fluorescent Labeling and Perfusion of Leukocytes (PMN or THP-1 for Human and RAW264.7 or Primary Mouse Monocytes for Murine Flow-Based Assay)

  1. Fluorescent labeling
    1. Label the leukocytes (1 x 106/mL) with the cell-permeant green fluorescent nucleic acid stain (1 µM). Incubate the cells for 30 min at 37 °C.
    2. Wash the cells with PBS by centrifugation at 300 x g for 5 min, and suspend cells to a concentration of 0.5 x 106 cells/mL (5 mL per test condition, depending on flow rate) in assay buffer.
  2. Flow chamber assay preparation
    1. For leukocyte adhesion on a cell monolayer, discard the cell culture medium from the dish, and assemble it into the flow chamber. Connect the tubing to the syringe. For leukocyte adhesion on immobilized platelets, connect the micro slide to the syringe. Pre-warm a water bath to 37 °C.
    2. Take a perfusion syringe (50 mL), install the syringe on the syringe holder, and set the pump on withdraw mode. Set the pump volume to 0 mL, and set the diameter to 26.70 mm for the 50 mL syringe.
    3. Connect an elbow luer connector to one end of the tubing. Place a luer lock coupler to the syringe and connect the tubing with the elbow luer connector to the luer lock coupler. Connect the free tubing end to the flow chamber.
  3. Leukocyte perfusion and adhesion
    1. For leukocyte adhesion on a cell monolayer, discard the cell culture medium from the dish, and assemble it into the flow chamber. Connect the tubing to the syringe. For leukocyte adhesion on immobilized platelets, connect the micro slide to the syringe.
    2. For leukocyte perfusion and adhesion over a vascular- or platelet monolayer, place the second tubing into a 50 mL conical tube containing assay buffer and fill the tubing with 1 mL pipet, then squeeze the tubing and connect it to a flow chamber.
    3. Prior to leukocyte perfusion, add 3 mM CaCl2 and 2 mM MgCl2 to the cell suspension and incubate for 5 min at 37 °C.
    4. Perfuse assay buffer prior to perfusion of cells to remove possible air from the chamber and tubing.
    5. Close tube ends by squeezing them tight and switching tubing from assay buffer to cell suspension, preventing air bubbles from being trapped. Perfuse cells with appropriate flow rate/shear stress until the first cells arrive.
    6. Perfuse cells with appropriate flow rate/shear stress for 2 min, and capture at least 6 pictures between 2–6 min of rolling and adherent cells with an inverted phase contrast/fluorescence microscope (e.g., EVOS-FL) using 100X magnification connected to a digital CCD or CMOS camera.
      NOTE: Flow rate/shear stress depends on flow chamber dimensions and viscosity of perfusate. For the perfusion chamber used here (see Table of Materials):
      τ=η*97.1*Φ
      τ: shear stress (1 dyn/cm2)
      η: viscosity (0.015 dyn*s/cm2)
      Φ: Flow rate (0.67 mL/min)
  4. Leukocyte de-adhesion
    1. For de-adhesion, switch tubing from cell suspension to assay buffer by squeezing the tubing, preventing air bubbles from becoming trapped.
    2. Detach cells by perfusion with assay buffer with an appropriate flow rate/shear stress (See 3.3.7 Note).
  5. Leukocyte transmigration
    1. For leukocyte transmigration, perfuse leukocytes (step 3) until an adequate quantity of leukocytes adheres (~50 cells/view field).
    2. Replace leukocyte suspension with assay buffer.
    3. Record transmigrating cells by time-lapse every 10–15 s for 30–60 min at 37 °C.

Disclosures

The authors have nothing to disclose.

Materials

Inverted fluorescence microscope e.g. EVOS-FL Life Technologies Europe bv
Pump e.g. model: 210-CE world precision instruments 78-9210W
Falcon 35 mm TC-Treated EasyGrip Style Cell Culture Dish corning 353001
50 mL syringe Becton Dickinson 300137
Silicone tubing VWR 228-0700
Elbow Luer Connector Male Ibidi 10802
Female Luer Lock Coupler Ibidi 10823
Flow chamber University Hospital RWTH Aachen Patent DE10328277A1: Baltus T, Dautzenberg R, Weber CPD. Strömungskammer zur in-vitroUntersuchung von physiologischen Vorgängen an Zelloberflächen. 2005.
Collagen Type I, rat tail Life Technologies Europe bv A1048301
Recombinant Human TNF-α Peprotech 300-01A
Thrombin Receptor Activator for Peptide 6 (TRAP – 6) Anaspec / Eurogentec 24191
SYTO 13 green fluorescent nucleic acid stain Life Technologies Europe bv S7575
Calcium chloride dihydrate Merck 102382
Magnesium chloride hexahydrate Sigma-Aldrich Chemie M2670
Mouse monocyte/macrophage RAW264.7 ATCC TIB-71
Dulbecco's Modified Eagle's Medium (DMEM), high glucose Gibco 11965092

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Cite This Article
A Laminar Flow-Based Assay to Study the Interactions Between Leukocytes and Endothelial Cells. J. Vis. Exp. (Pending Publication), e21922, doi: (2024).

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