The use of flow cytometry to assess neutrophil infiltration in the injured murine spinal cord.

Inflammatory cells, including neutrophils, are likely candidates in promoting early cell death after spinal cord injury. We describe a simple and reliable method for obtaining neutrophils from the injured murine spinal cord for flow cytometric quantification. Mice were subjected to either a moderate or severe spinal cord contusion injury and euthanized 24 h later. The area of maximal damage, designated the epicenter, was prepared for assessment of myeloperoxidase (MPO) activity, quantitative immunocytochemistry, or quantification of immunolabeled neutrophils by flow cytometry. For flow cytometry, a cell suspension was prepared from the epicenter by gentle mechanical disruption. After centrifugation, the pellet was resuspended, immunolabeled for neutrophils, and analyzed. There was no detectable MPO activity in the injured spinal cord. In contrast, neutrophil infiltration was confirmed by immunocytochemistry and found to be significantly greater in the more severely injured group. Flow cytometry, using a standard neutrophil marker, revealed a similar significant increase in immunolabeled cells in the more severely injured group. However, when cell viability was determined in the neutrophil labeled population, no significant difference in the numbers of live neutrophils were noted between the two injured groups. Together, these findings demonstrate an effective method for the detection and quantification of viable neutrophils in the injured murine spinal cord.