Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers.
High-pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze-substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high-pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high-pressure freeze, freeze-substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high-pressure freezing, freeze-substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.
Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands. N.Jimenez-Gil@bio.uu.nl
N
N
Jiménez
B M
BM
Humbel
E
E
van Donselaar
A J
AJ
Verkleij
K N J
KN
Burger
D000818:Animals; D018938:Caco-2 Cells; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods*; D017110:Freeze Substitution; D006367:HeLa Cells; D006801:Humans; D011312:Pressure