Immune cell subpopulations of tissue fluid were quantified by flow cytometry. (A) At 48 h, increased expression of markers, such as, PMN cell marker (stained using anti-PMN-FITC), and β-endorphin marker (stained using anti-β-endorphin-PE), on leukocytes is observed in G-CSF–treated rats with CCI than in vehicle-treated rats with CCI (n = 6 per group). CD45-PE-Cy5 and ED2-PE antibodies were used to react with all hematopoietic cells and macrophages, respectively. Low staining with the respective control antibodies, confirmed a specific antibody staining of opioid-containing PMN cells. In contrast, low staining with ED2-PE and PMN-FITC antibodies (0.3–3.4%), confirmed a specific staining of PMN cells by PMN-FITC antibody. (B) At 48 h, the number of β-endorphin–containing PMN cells in G-CSF–treated rats with CCI is significantly higher than that in vehicle-treated rats with CCI. Data are shown as the mean±SEM, n = 6 per group; t test, *p<0.05.