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) Fluorescent labeling 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. 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 t…
Divulgazioni
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 |
Tags
Citazione di questo articolo
A Laminar Flow-Based Assay to Study the Interactions Between Leukocytes and Endothelial Cells. J. Vis. Exp. (Pending Publication), e21922, doi: (2024).