A technique for the isolation of submucosal gland cells from cat trachea

We have developed a procedure to isolate submucosal gland cells from cat trachea. The excised trachea was stripped of surface epithelium by stroking the luminal surface with a nylon brush. The remaining submucosa was scraped free from underlying cartilage and minced into small fragments. To disperse glandular cells from these fragments, we subjected the minced tissue to both enzymatic (collagenase and elastase) and mechanical treatment. In 23 preparations of cells we yielded an average (+/- SE) of 8.4 +/- 0.9 (X 10(6] cells. In eight cell preparations 95 +/- 1% of the cells stained with periodic acid-Schiff stain, suggesting that the cells are of glandular origin. We used the following criteria to assess cell viability. The dye trypan blue was excluded by 92 +/- 1% of the cells (n = 23). Under the electron microscope, cellular membranes and organelles appeared normal. The isolated cells consumed oxygen at an average rate of 1.34 +/- 0.05 microliters O2 X h -1 X (10(6) cell) -1, (n = 65). Oxygen consumption was constant for at least 4 h after cell isolation, was inhibited 21% by 10(-4) M ouabain, and was subsequently stimulated to 135% above basal levels by 4 X 10(-5) M dinitrophenol.