Methods Methods Assays of apoptotic

							Methods




Assays of apoptotic cell death on H9C2 cell line by flow cytometry:




    The H9C2 cell line was cultured at 1 x 105 cells/mL in IMDM containing 5%


FCS, and pretreated with TPO (100 ng/mL) for 2 hr before the addition of DOX (0.58


µg/mL) for 24 hr. The Annexin V: FITC/propidium iodide (PI) Apoptosis Detection


Kit (BD Pharmingen, San Diego, CA, USA) was used according to the


manufacturer’s instruction. The proportions of apoptotic cells (Annexin V positive


and PI negative) and total dead cells (Annexin V positive) were analyzed by flow


cytometry using the LYSIS II software (FACScan, BD Pharmingen). Ten thousand


events were acquired for each sample. For the expression of Caspase-3, the cells were


treated with 500 µL 1 x FACS Permeabilizing Solution (BD Pharmingen) for 10 min


in the dark at room temperature. After washing, they were stained with 10 µL of


phycoerythrin-conjugated antibody against active Caspase-3 (BD Pharmingen) for 30


min before flow cytometry analysis. Mitochondrial integrity in H9C2 cells were


monitored by the JC-1


(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine) reagent kit (BD


ApoAlert Mitochondrial Membrane Sensor Kit; BD Biosciences, Palo Alto, CA,
USA). Mitochondria with normal m would concentrate JC-1 into aggregates

(red/orange fluorescence in FL2) while in depolarized mitochondria, JC-1 forms


monomers (green fluorescence in FL1). For this assay, the H9C2 cell line was


pretreated with TPO (100 ng/mL) for 2 hr before the addition of DOX for 2 hr. The


cells were washed to remove DOX and fresh medium containing TPO was added for


another 22 hr. H9C2 cells were trypsinized (0.25%, Gibco), washed with PBS and


resuspended in 1 mL BD MitoSensor Reagent for 15 min at 37°C in a 5% CO2


incubator. The cells were washed with 1 mL incubation Buffer and analyzed by flow


cytometry.




Primary neonatal rat cardiomyocyte culture and beating rate measurement:




     Heart ventricles were dissected from neonatal Sprague-Dawley rats at day 1 after


birth. These tissues , after washing with phosphate buffered saline (PBS), were


digested in 2 ml collagenase (200 U/mL; Gibco) and 1 mL Dulbecco’s modified


Eagle’s medium (DMEM) for 1 hr at 37oC. After centrifugation at 400 x g for 7 min,


the cell pellet was suspended in DMEM and Medium 199 in a ratio of 4:1, 5% FCS


and 10% horse serum.18 The majority of fibroblasts was removed by attachment for 1


hr. Non-attached cells were plated in a 75 cm2 flask at 37oC for 1 hr. Enriched
myocytes were cultured in 24-well plates at 2x105 cells/cm2 with daily medium


change. Treatment studies were performed after myocytes were established for 5-6


days when the cells were beating at relatively steady rates. The beating rates and


morphology of cardiomyocytes were video-captured on CompactFlash memory cards


in a digital camera connecting to a phase contrast inverted microscope. Spontaneous


beatings of each colony of cells were recorded for 40 sec. The beating cells were


treated with a single dose of DOX (1 M or 0.58 g/mL) with and without

pretreatment (1 hr before) with TPO (50 or 100 ng/mL) or dexrazoxane (5.8 g/mL).

The culture medium was replaced with fresh TPO and dexrazoxane daily. Beating


rates of each colony of cells were measured at 24 hr and 48 hr post-DOX treatment


and expressed as the percentage of those measured before the addition of any drug.


Results are expressed as the beating rate (percentage) relative to that of the same


colony of cells at zero time.




TUNEL assay on heart cells from experimental animals:




     A pilot study demonstrated that DOX-induced apoptotic cells were most


noticeable by the TUNEL assay on Day 3 post-DOX treatment. Formaldehyde-fixed


heart sections (4-m) were processed with the In Situ Cell Death Detection Kit,
Fluorescein (Roche Diagnostics GmbH, Penzberg, Germany) according to the


manufacturer’s instruction. Six sections from each heart were examined under a Zeiss


Axioplan 2 microscope linked to a SPOT-Cooled Color Digital Camera (Diagnostic


Instruments, Sterling Heights, MI, USA). All TUNEL positive nuclei from


cardiomyocytes were counted in each section and the section area was computed by


SPOT Advanced Software (Version 4.1.3, Diagnostic Instruments Inc.). Results are


presented as the number of apoptotic cells per unit area (mm2 ).