Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections.
Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented. The cells 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 medium 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 method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.
Division of Physiology, UMDS St Thomas' Hospital Campus, London, U.K.
A
A
Warley
K P
KP
Cracknell
H B
HB
Cammish
C H
CH
Twort
J P
JP
Ward
S J
SJ
Hirst
D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000032:analysis; D016616:Cryoultramicrotomy; D004577:Electron Probe Microanalysis; D008274:Magnesium / Q000032:analysis; D008297:Male; D009130:Muscle, Smooth / Q000737:chemistry* / Q000648:ultrastructure; D010042:Ouabain / Q000494:pharmacology; D010758:Phosphorus / Q000032:analysis; D011188:Potassium / Q000032:analysis; D011817:Rabbits; D012964:Sodium / Q000032:analysis; D014132:Trachea / Q000737:chemistry* / Q000648:ultrastructure
D002712:Chlorides; D010758:Phosphorus; D010042:Ouabain; D012964:Sodium; D008274:Magnesium; D011188:Potassium