Diff quik blood stain

Two days after infection, lungs of mice were lavaged after the cannulation of trachea with polyethylene tubing, which was attached on a tuberculin syringe. The same procedure was applied to sham-infected (submitted to surgical stress and injected with 50 μL of PBS) and normal mice of both mouse strains. The lungs were lavaged by repeated injections of 0.5 mL of sterile PBS (final volume, 2.0 mL). The recovered fluid was cytospun at 1,200 rpm (Shandon Cytospin, Pittsburgh, PA) onto glass slides and stained by the Diff-Quik blood stain (Baxter Scientific, Miami, Fla); the supernatant was removed and cells were analyzed for leukocyte subsets. The frequency of polymorphonuclear neutrophils, lymphocytes, and macrophages were morphologically evaluated, and the absolute cell number was calculated based on the total cell counts of BALF.

For differential counts, samples of lung cell suspensions were cytospin (Shandon Cytospin) onto glass slides and stained by the Diff-Quik blood stain (Baxter Scientific). A total of 200 to 400 cells were counted from each sample. The absolute number of a leukocyte subset was calculated by multiplying the percentage of each subset in an individual sample by the total number of lung leukocytes in that mouse.