Home › Immunology › Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
Immunology JoVE (Open Access) Citable · DOI

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method

DOI: 10.3791/2698-v
What you'll learn
  • ✓ Prepare injured spinal cord tissue for flow cytometric analysis via enzymatic dissociation
  • ✓ Remove myelin/lipid debris using OptiPrep density gradient centrifugation
  • ✓ Perform immunolabeling and quantify immune cell populations by flow cytometry
  • ✓ Improve detection sensitivity and accuracy of cellular inflammation in CNS tissue
Protocol

Biopharma Insights Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Difficulty
advanced
Total time
~4–6 hours per spinal cord sample
Model organism
Mouse (strain not specified)
Biosafety
BSL-1

Steps

1
Dissociate injured spinal cord tissue

Enzymatically dissociate spinal cord tissue to generate single-cell suspension. This step separates cells from intact tissue matrix to enable downstream flow cytometric analysis.

▶ 01:23
2
Prepare OptiPrep gradient and separate debris

Create OptiPrep density gradient solutions and perform centrifugation-based separation to remove myelin and lipid debris from the cell suspension, improving signal-to-noise ratio.

▶ 02:47
3
Immunolabel and analyze immune cells by flow

Apply fluorescent antibodies to label specific immune cell populations, then acquire and analyze samples by flow cytometry to quantify cellular inflammation.

▶ 04:27
4
Validate debris removal enhances detection

Compare flow cytometric data before and after myelin debris removal to demonstrate improved sensitivity and accuracy in quantifying immune cells in injured spinal cord.

▶ 06:16
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