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

Protocollo

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.

Materiali

Name	Company	Catalog Number	Comments
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