“…To measure T and B cell frequency and proliferation, peripheral blood mononuclear cells (PBMC) and BAL cells were first stained with anti-CD4 (Tonbo Biosciences, San Diego, CA), CD8b (Beckman Coulter, Brea, CA), CD20 (BioLegend, San Diego, CA), IgD (Southern Biotech, Birmingham, AL), and CD27 (Tonbo Biosciences), then fixed and permeabilized before staining with anti-Ki67 (BD Pharmingen, San Diego, CA). To determine the frequency of MAC-specific T cells, 10 6 PBMC and BAL cells were stimulated with 10 ml MAC lysate, anti-CD3 (positive control), or media alone (negative control) for 16 hours in the presence of brefeldin A. Cells were stained with anti-CD4 and CD8b, then permeabilized and intracellularly stained with anti-IFNg, TNF-a, and macrophage inflammatory protein-1 beta (MIP1b) (15).…”