Detection of infiltrated macrophage subtypes in injured spinal cord of rats by flow cytometry

Objective: To develop the technique for measuring infiltrated macrophage subtypes in injured spinal cord of rats by flow cytometry. Methods: Contusive spinal cord injury( SCI) model in five healthy female SD rats( SCI group) were established by New York University Impactor， other five healthy female SD rats were treated with sham operation( sham operation group) . The injured spinal cords were collected and made into single cell suspension at day 7 after SCI. The infiltrated macrophages were concentrated by density gradient centrifugation using Percoll. The type M1 and M2 macrophages in lesions were identified using CD68 and CD86，or CD163 fluorescent antibodies， and measured by flow cytometry. Results: In rat's spinal cord single cell suspension of the sham operation group，the percentage of CD68 + cells in total cells was( 0. 07 0. 02) %， the percentages of CD86 + and CD163 + cells in CD68 + cells were (9. 90 3. 28) % and( 4. 95 0. 17) %， respectively. In rat's spinal cord single cell suspension of the SCI group， the percentage of CD68 + cells in total cells was( 0. 21 0. 09) %， the percentages of CD86 + and CD163 + cells in CD68 + cells were( 18. 71 0. 49) % and( 27. 07 3. 74) %，respectively. These differences between the sham operation group and SCI group were statistical signficance ( P ＜ 0. 05 to P ＜ 0. 01) . Conclusions: By using the mouse anti-rat CD68，CD86 and CD163 fluorescent antibodies， the infiltrated M1 and M2 macrophage subtypes can be successfully detected by flow cytometry.