PREPARATION OF CULTURED AIRWAY SMOOTH-MUSCLE FOR STUDY OF INTRACELLULAR ELEMENT CONCENTRATIONS BY X-RAY-MICROANALYSIS - COMPARISON OF WHOLECELLS WITH CRYOSECTIONS
A. Warley et al., PREPARATION OF CULTURED AIRWAY SMOOTH-MUSCLE FOR STUDY OF INTRACELLULAR ELEMENT CONCENTRATIONS BY X-RAY-MICROANALYSIS - COMPARISON OF WHOLECELLS WITH CRYOSECTIONS, Journal of Microscopy, 175, 1994, pp. 143-153
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.