Figure 5

Inhibition of oxidative burst response by KO alveolar leukocytes. Alveolar leukocytes (0.5 × 106 cells) were stained with F4/80-Cy-Chrome and Gr1-APC for 30 min on ice, washed in PBS, warmed up at 370C for 5 min and loaded with 5mM dihydrorhodamine 123 (Molecular Probes, Eugene, OR). After 10 min at 370C, cells were split in two equal aliquots, and PMA (Sigma, St. Louis, MO) was added to one aliquot at final concentration of 1mM. After 10 min incubation cells were washed in ice-cold PBS and immediately subjected to FACS analysis. Cells were gated on neutrophils (Gr1hi), or monocyte/macrophages (F4/80+) and percentage of cells positive for dihydrorhodamine 123 fluorescence with or without PMA treatment was determined for each gate. Results shown are mean of 3 independent experiments ± SEM. (n=5/group). * denotes p value<0.05 compared to WT without PMA treatment and # denotes p value<0.05 compared to WT post-PMA treatment. While WT cells respond to PMA before as well as after OVA challenge, cells from both KO mice before as well as after OVA, failed to respond appreciably. DHR was measured at Fluorescent channel 1 in using a BD Facscaliber and DHR+ cells (CD45+gated and Gr-1+ gated or F4/80+ gated) were analyzed using CellQuestpro software.