Biomedical Engineering Reference
In-Depth Information
2.5 Northern Blot Analysis
Cells were homogenized in Trizol reagent (Gibco) and purified according to the
manufacturers instructions. After phenol-chloroform extraction and sodiumace-tate
precipitation RNA samples were quantitated by spectrophotometry at 260 nm. After
seperation on an agarose gel (15 ȝg/lane) the RNA was transferred to a nylon
membrane (Hybond-N, Amersham) and UV-crosslinked. After prehybridization for 1
h at 65°C the membrane was incubated with DIG-labeled (Boeh-ringer Mannheim
DIG RNA labelling kits SP6/T7) oligonucleotides of Cx-40 (372 bp) or cardiac
troponin-I (TnI, 195 bp) at 68°C over night. Amounts of the RNA samples were
confirmed by additionally running each blot of Cx-40 and TnI with
ȕ
-actin (304 bp)
nucleotides. The signals were visualized using Į-DIG-AP (1:10,000, Roche) in 1%
block reagent and CDP-Star reagent (Boehringer Mannheim). Neonatal heart tissue
and skeletal muscle served as positive and negative control group.
2.6 Quantification of Northern Blot Signals
The blots were scanned (Sharp JX-330). Quantification was performed by measuring
the optical density (pixel and area) of the blot signals with the ''Enhanced analysis
system'' for gel documentation (Herolab GmbH, Wiesloch, Germany). To normalize
the amount of RNA/lane optical density values for Cx-40 and troponin I of different
groups (group 1 treated with NRG-1-Į, group 2 NRG-1-
ȕ
, group 3 control group)
were divided by the corresponding optical density of
ȕ
-actin. To normalize variability
in the proportion of cardiomyocytes in different cell samples, the resulting values for
Cx-40 (Cx-40/
ȕ
-actin) were divided by the values for cardiac specific troponin I
(troponin I/
ȕ
-actin). All experiments were repeated at least three times.
2.7 Flowcytometry
Due to small amounts of membrane protein flowcytometry rather than western
blotting was performed to evaluate protein expression. For identification of car-
diomyocytes, cells were stained with a mouse IgG1 monoclonal antibody EA-53 to
sarcomeric Į-actinin (diluted 1:800, Sigma) using the BD Cytofix/Cytoperm Kit (BD
Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. Co-
staining was performed using the following primary antibodies: a rabbit polyclonal
antibody to mouse Cx-40 (diluted 1:100), a rabbit polyclonal antibody to mouse Cx-
43 (diluted 1:25), or a rabbit polyclonal antibody to Cx-45 (diluted 1:50), respectively
(all connexin antibodies from Chemicon, Temecula, CA, USA).
After incubation with indodicarbocyanin (Cy5) labeled donkey anti mouse IgG
(diluted 1:500, Dianova, Hamburg, Germany) and carbocyanin (Cy2) labeled goat
anti rabbit IgG (1:100, Dianova) as secondary antibodies, Į-actinin positive
cardiomyocytes were analyzed for connexin expression by flowcytometry using a
FACSCalibur instrument (BD Biosciences, Heidelberg, Germany). Data were further
processed using WinMDI 2.8 software, MFI (Martz, Eric. 1992-2001.
http://www.umass.edu/microbio/mfi), and GraphPad Prism (version 3.02 for
Windows, GraphPad Software, San Diego, CA, USA). Cardiomyocytes incubated
with isotype control antibodies or rabbit serum served as negative controls.
