Immunology JoVE (Open Access) Citable · DOI

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Name: Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Uploaded: 2026-06-23
Duration: 12 min 14 s
Description: Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

DOI: 10.3791/53658-v

What you'll learn

✓ Isolate viable immune cells from ischemic mouse brain tissue using perfusion and enzymatic dissociation
✓ Remove myelin and debris using density gradient separation to enrich leukocyte populations
✓ Perform flow cytometric phenotyping and absolute quantification of infiltrating immune cell subsets

Protocol

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Difficulty

advanced

Total time

~3–4 hours per mouse (transcardial perfusion through flow cytometry analysis)

Model organism

Mouse (ischemic stroke model)

Biosafety

BSL-1

Steps

Perform transcardial perfusion and brain dissection

Anesthetize mouse and conduct transcardial perfusion with saline to remove blood cells, then rapidly extract and dissect the ischemic brain hemisphere.

▶ 01:07

Dissociate cerebral tissue enzymatically

Mechanically and enzymatically dissociate the ischemic brain tissue into a single-cell suspension using papain and trituration.

▶ 04:24

Remove myelin and cell debris by density gradient

Apply density gradient separation using Percoll or similar medium to enrich leukocytes and deplete myelin and dead cell material.

▶ 06:21

Perform flow cytometry immunostaining and quantification

Stain isolated cells with fluorescently-labeled antibodies, acquire data on flow cytometer, and use counting beads for absolute cell quantification.

▶ 07:34

Analyze and interpret infiltrating immune cell data

Review representative flow cytometry plots and histograms showing phenotypic identification of infiltrating macrophages, microglia, and lymphocytes in ischemic brain.

▶ 09:14

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