Purification of Murine Pulmonary Type II Cells for Flow Cytometric Cell Cycle Analysis

Abstract

Mice are widely used as animal models for in vivo lung disease. Despite this fact, few methods exist for isolation of type II pneumocytes from mouse lung, limiting the study of alveolar epithelial characteristics in these models. This study investigated several methods for labeling murine lung cell suspensions for flow cytometric identification and sorting of type II pneumocytes. Crude lung cell suspensions were prepared after intratracheal instillation of Dispose and were labeled using phosphine alone or in combination with Helix pomatia lectin, Madura pomifera lectin, or anti-murine-CD32. Crude cell suspensions yielded 17.4 million cells per animal with 19.5% type II pneumocytes by Pap staining. Ultrastructural evaluation of the sorted cell pellets (1—1.5 million cells each) demonstrated optimal type II cell purity in preparations labeled with phosphine and anti-CD32 (94.3% type II cells, 0.4% macrophages, 2.8% Clara cells, and 2.5% other). Nuclear suspensions appropriate for cell cycle analysis were produced by sorting the type II cells directly into hypotonic propidium iodide, and these preparations clearly demonstrated a substantial increase in S-phase type II cells during proliferative repair of BHT-induced acute lung injury.

Key Words:

• pneumocytes
• type II cells
• mouse
• flow cytometry
• cell cycle