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In-Depth Information
Apoptosis Assays
Apoptosis assessment was performed using Annexin-V-FLUOS (fluorescein)/pro-
pidium iodide (PI) staining kit (Roche Diagnostics GmbH, Penzberg, Germany).
The samples were prepared according to the manufacturer's instructions and were
analyzed by flow cytometry. Annexin-V-positive and PI-negative cells were scored
as apoptotic cells.
Apoptotic morphological changes were evaluated by DAPI (4',6'-diamidino-2-
phenylindole) staining. After oxidative stress induction, cells were gently washed
with phosphate-buffered saline (PBS) and fixed with cold methanol for 10 min-
utes. The plates were washed twice with PBS and incubated for 5 minutes with
300
µ
M DAPI (catalogue number D-3571, Molecular Probes, Eugene, Oregon,
USA) at 37°C. Finally, the cells were washed three times with PBS and examined
by fluorescence microscopy. Apoptotic cells were identified by condensation and
fragmentation of the nuclei, and were quantified by counting a total of 200 nuclei
from each well and calculating the percentage of apoptotic nuclei.
rat Model of Acute Myocardial infarction
Male Sprague-Dawley rats (approximately 250 g; Harlan Lab, Jerusalem, Israel)
were subjected to acute myocardial infarction (AMI) induction by permanent
coronary artery occlusion, as previously described [19].
RhEPO-transduced and non-transduced fibroblast transplantations were per-
formed 7 days after AMI. After 10 days in culture (7 days after transduction, 3
passages), cardiac fibroblasts were prepared for transplantation by trypsinization
with 0.25% Trypsin-EDTA solution for 1 minute at 37°C. The rhEPO expression
level in prepared cells was 7.0 ± 0.23 mIU/24 h/106 cells. Following centrifuga-
tion (1,500 rpm, 10 minutes at room temperature) the cells were re-suspended in
saline. The final cell density for implantation was 1 × 106 cells/100
µ
l. Rats were
anesthetized and the chest was opened under sterile conditions. The infarcted
area was identified visually by the surface scar and wall motion akinesis. Rats were
randomized and selected either for injection of 1 × 106 rhEPO-transduced cells,
1 × 106 non-transduced cells, or saline (n = 5 in each group). All injections were
made into the center of the scar. After the injection, the air was expelled from the
chest, and the surgical incision was sutured closed [19,20].
echocardiography
Transthoracic echocardiography was performed on all animals before transplan-
tation (baseline echocardiogram) and 5 and 9 weeks later (4 and 8 weeks post-
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