PREPARATION OF CULTURED AIRWAY SMOOTH-MUSCLE FOR STUDY OF INTRACELLULAR ELEMENT CONCENTRATIONS BY X-RAY-MICROANALYSIS - COMPARISON OF WHOLECELLS WITH CRYOSECTIONS

Methods for growing and preparing smooth muscle cells, isolated from r abbit trachealis, for X-ray microanalysis studies are presented. The c ells are grown on Pioloform-covered gold grids supported on Thermanox coverslips. This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies . The cells are analysed as whole mounts after removal of growth mediu m by washing, followed by cryofixation and freeze drying. The effects of different washing media (0.3 M sucrose, 0.15 M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A me thod for growing the cells as monolayers and mounting the cryofixed mo nolayers for cryosectioning is also given. Comparison of elemental con centrations in the cytoplasm of distilled-water washed cells with thos e of the cytoplasm of cryosectioned cells obtained from the same anima l showed good agreement between values obtained from the two preparati ve procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be impor tant in understanding the mechanisms of proliferation which lead to in creased airway smooth muscle mass in persistent severe asthma.