Biomedical Engineering Reference
In-Depth Information
Fig. 2
Localization of CFTR and STX16 in T84 monolayers. Colonic T84 epithelial
cells were cultured in permeable supports to form polarized monolayers, and
exogenous CFTR was expressed by transient transfection with pCMV-
CFTR. Fluorescent images of the vertical
z
axis section were obtained after stain-
ing with anti-STX16 (
red
) and anti-CFTR (
green
) using a Zeiss LSM 510 confocal
microscope. Fluorescence intensities were analyzed with the MetaMorph soft-
ware. Note that
red
and
green colors
are highly co-localized at subapical regions
(
yellow
). Reprinted by permission from the American Society for Biochemistry
and Molecular Biology, reprinted from Gee et al. [
3
]
endogenously or exogenously expressed CFTR in the apical plasma
membrane of T84 and CFPAC-1 cells can be quantifi ed using a
Z
-axis scan of a polarized cell sheet after proper membrane culture
and immunostaining (Fig.
2
). In addition, immunohistochemistry
using tissue samples from gene-manipulated animals can provide
information related to the in vivo effects of a gene of interest on
membrane traffi cking. As shown in Fig.
3
, mice expressing trans-
genic GRASP55 showed improved expression of
ʔ
F508-CFTR at
the apical membrane in colonic crypt cells [
3
].
1. HEK293 cells cultured on coverslips or epithelial cells cultured
on permeable supports can be used for immunostaining
36-48 h after transfection with plasmids or recombinant
viruses for expressing CFTR or other proteins of interest.
2. Fix cells grown on coverslips (or permeable supports) with
3.7 % formaldehyde for 10 min and permeabilize the cell mem-
brane with 0.1 % Triton X-100 for 10 min. Alternatively, 100 %
methanol (−20 °C freezer chilled) can be used for both fi xation
and permeabilization simultaneously (
see
Note 1
).
3.2 Immuno-
cytochemistry
