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Cell Biology JoVE (Open Access) Citable · DOI

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

DOI: 10.3791/2316-v
What you'll learn
  • ✓ Isolate primary pulmonary endothelial cells from neonatal mouse lungs
  • ✓ Perform magnetic bead-based purification using PECAM-1 and ICAM-2 antibodies
  • ✓ Evaluate endothelial cell purity and viability post-isolation
Protocol

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Difficulty
advanced
Total time
~3–4 hours per neonatal mouse
Model organism
Mouse (neonatal)
Biosafety
BSL-1

Steps

1
Prepare anti-PECAM-1 antibody-conjugated magnetic beads

Set up magnetic bead conjugation with anti-PECAM-1 antibodies according to manufacturer protocol. This preparation step ensures beads are ready for the subsequent cell isolation and sorting phases.

▶ 00:57
2
Isolate mouse pulmonary endothelial cells from neonatal lung

Perform mechanical and enzymatic tissue dissociation of neonatal mouse lungs to generate a single-cell suspension. Apply the first magnetic purification step using anti-PECAM-1 beads to enrich endothelial cells.

▶ 03:36
3
Sort lung endothelial cells with anti-ICAM-2 Dynabeads

Apply second-stage magnetic purification using anti-ICAM-2 conjugated Dynabeads to further enrich microvascular endothelial cells and remove contaminating populations.

▶ 06:56
4
Evaluate purity and viability of isolated endothelial cells

Perform flow cytometry or immunofluorescence staining to assess endothelial cell identity, purity, and viability of the final isolated population.

▶ 09:13
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