Home › Biochemistry › Human Neutrophil Flow Chamber Adhesion Assay
Biochemistry JoVE (Open Access) Citable · DOI

Human Neutrophil Flow Chamber Adhesion Assay

DOI: 10.3791/51410-v
What you'll learn
  • ✓ Prepare HUVEC monolayers and purified ligand substrates for adhesion assays
  • ✓ Isolate human neutrophils from peripheral blood using density separation
  • ✓ Quantify neutrophil adhesion under physiological shear stress conditions
  • ✓ Compare neutrophil adhesion to endothelial cells versus purified adhesion molecules
Protocol

Biopharma Insights A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Difficulty
advanced
Total time
~4–6 hours per experiment (including cell isolation and assay running time)
Model organism
Human peripheral blood neutrophils; HUVEC (human umbilical vein endothelial cells)
Biosafety
BSL-2

Steps

1
Prepare HUVEC monolayer on flow chamber

Culture and plate human umbilical vein endothelial cells on the inner surface of a flow chamber to establish a confluent endothelial substrate. This mimics the physiological environment of blood vessel walls.

▶ 01:50
2
Coat flow chamber with purified adhesion ligands

Apply purified adhesion molecules (e.g., selectins, integrins) to the flow chamber surface as an alternative substrate. This allows isolation of specific adhesion receptor–ligand interactions.

▶ 03:07
3
Isolate neutrophils from human peripheral blood

Separate neutrophils from whole blood using density gradient centrifugation or similar methods to obtain a purified neutrophil population for the adhesion assay.

▶ 05:11
4
Execute flow chamber adhesion assay under shear stress

Perfuse the isolated neutrophils through the flow chamber under controlled shear stress conditions to quantify adhesion to either HUVEC monolayers or purified ligands. Measure neutrophil attachment and rolling dynamics.

▶ 08:33
5
Analyze adhesion results and compare substrates

Quantify neutrophil adhesion data across experimental conditions (HUVEC versus purified ligands) and assess adhesive properties including rolling, firm adhesion, and transmigration rates.

▶ 10:20
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