Home › Biochemistry › A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress
Biochemistry JoVE (Open Access) Citable · DOI

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

DOI: 10.3791/51330-v
What you'll learn
  • ✓ Prepare human hepatic sinusoidal endothelial cells with cytokine stimulation for adhesion assays
  • ✓ Isolate and treat peripheral blood lymphocytes for flow-based recruitment studies
  • ✓ Execute a flow adhesion assay under physiological shear stress conditions
  • ✓ Analyze and quantify leucocyte adhesion dynamics to endothelial monolayers
Protocol

Biopharma Insights Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Difficulty
advanced
Total time
~4-6 hours per assay (cell stimulation, lymphocyte isolation, flow setup, and recording)
Model organism
Human primary hepatic sinusoidal endothelial cells; human peripheral blood lymphocytes
Biosafety
BSL-1

Steps

1
Stimulate endothelial cells with cytokines

Treat cultured human hepatic sinusoidal endothelial cells with appropriate cytokines to induce adhesion molecule expression and prepare the endothelial monolayer for leucocyte recruitment assays.

▶ 01:59
2
Isolate and pretreat peripheral blood lymphocytes

Extract lymphocytes from human peripheral blood and apply necessary pretreatment protocols to prepare cells for adhesion assay conditions.

▶ 04:11
3
Configure flow adhesion assay apparatus

Assemble the flow system components including flow chamber, microscopy setup, and shear stress delivery to create physiological conditions for leucocyte recruitment.

▶ 06:09
4
Execute flow assay and record adhesion events

Perform the flow adhesion assay using phase contrast microscopy while maintaining physiological shear stress, capturing real-time leucocyte adhesion and recruitment to the endothelial monolayer.

▶ 08:33
5
Analyze adhesion cascade dynamics and outcomes

Review recorded adhesion events to quantify and characterize the sequential steps of the leucocyte adhesion cascade across hepatic sinusoidal endothelium.

▶ 09:35
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