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Immunology JoVE (Open Access) Citable · DOI

Quantitation of Endothelial Cell Adhesiveness In Vitro

DOI: 10.3791/52924-v
What you'll learn
  • ✓ Prepare endothelial cell monolayers on glass coverslips for adhesion assays
  • ✓ Isolate and prepare monocytes for endothelial cell adhesion testing
  • ✓ Quantify adhesive endothelial cells using enumeration methodology
  • ✓ Interpret adhesiveness data from in vitro cell-cell interaction assays
Protocol

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

Difficulty
intermediate
Total time
~4–6 hours per assay (cell preparation through quantification)
Model organism
Human endothelial cells (in vitro); human monocytes (in vitro)
Biosafety
BSL-1

Steps

1
Prepare glass coverslips for cell culture

Clean and prepare glass coverslips to serve as substrate for endothelial cell monolayer growth. Proper preparation ensures optimal cell adhesion and monolayer formation.

▶ 01:31
2
Culture endothelial cells into confluent monolayer

Seed endothelial cells on prepared coverslips and maintain culture conditions until a complete monolayer is established. This monolayer serves as the adhesion substrate.

▶ 02:41
3
Isolate and prepare monocytes for adhesion assay

Extract and process monocytes to obtain a viable, uniform cell suspension at defined concentration. These cells will be used to assess endothelial adhesiveness.

▶ 04:18
4
Enumerate adhesive endothelial cells under microscopy

Apply monocytes to endothelial monolayer, allow adhesion, then systematically count and quantify the number of monocytes bound to endothelial cells across defined fields.

▶ 06:50
5
Analyze quantified adhesiveness data and results

Interpret enumeration counts to determine endothelial cell adhesive capacity. Present quantitative metrics reflecting the degree of monocyte–endothelial cell interaction.

▶ 07:50
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