These cells were collected, stained with CD11c-PE, a cell surface marker of murine dendritic cells and at least 10,000 cells, based on forward and 90° light scatter properties, were analyzed by flow cytometry.
Following incubation, the cells were collected, washed and stained for cell surface markers (CD3,CD4) followed by intracellular cytokine staining (IFNγ, IL-4) and at least 50,000 live cells, based on forward and 90° light scatter properties, were analyzed by flow cytometry as described above.
The CD34+, CD34+CD38-, and CD34+c- kit+ hematopoietic progenitor populations were quantitated from the gated MNC population (lymphocytes and monocytes, based on forward versus side light scatter) using the FlowJo™ software program (Tree Star, Inc., Ashland, OR, USA); backgating demonstrated that all stem/progenitor subpopulations were within the gated MNC population.
Gating of CD4+ and CD8+ cells in the lymphocyte-rich regions was based on forward-angle light scatter and log Phycoerythrin fluorescence (FL2-H) and then the expression of CD62L (FL1-H) and CD44 (FL4-H) on CD4+ or CD8+ gated lymphocytes were analyzed.
BAL cell subpopulations were identified in the pre- (0 h) and post-challenge (4 h, 24 h, 48 h) BAL samples, with gating based on forward- and side- light scatter (FSC, SSC) properties as reported elsewhere [ 23].
Previous known decidable classes were based on forward chaining.
Neutrophils were gated based on forward and side scatter.
Cells were gated based on forward and side scatter.
Based on forward selection, age was the only significant variable.
