Tsuji et. al. amniotic membrane-derived stem cell (2009/205260-R3 ...

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
digestion was filtered (120-µm mesh) and centrifuged (400 rpm for 3
minutes). The cells were then stored in a Tyrode!s solution
supplemented with 0.5 mmol/L CaCl 2 and 10 mmol/L of BDM at room
temperature. Tyrode!s solution contained (mmol/L) NaCl 129.5, KCl 5,
NaH 2 PO 4 0.9, NaHCO 3 20, MgSO 4 1.2, CaCl 2 1.8, and glucose 5.5 (pH
adjusted to 7.4 at the 37°C) with oxygenation (0 2 95% - CO 2 5%).
Nominally Ca 2+ -free Tyrode!s solution was prepared by simply omitting
the CaCl 2 .
The obtained cardiomyocytes were mounted on the inverted fluorescence
microscope (IX71, Olympus, Tokyo, JAPAN). Rod-shaped cells with
strong EGFP fluorescence intensity were searched in the 4x objective
lens and confirmed by phase contrast microscope that the cells have a
clear striation, which then defined them as EGFP-positive
cardiomyocytes. The EGFP-positive cardiomyocytes were picked up by
pipette and stored in the fresh Tyrode!s solution to increase the
concentration of EGFP-positive cardiomyocytes. After every EGFPpositive
cardiomyocyte was separated into a new dish of fresh Tyrode!s
solution, we picked up EGFP-positive cardiomyocytes again to discard
contaminated EGFP-negative cardiomyocytes by glass pipette (tip
diameter was about 50~100 µm) mounted on a micromanipulator (WR-6,
Narishige, Tokyo, Japan). We performed this step again and we finally
obtained 100% EGFP-positive cardiomyocytes.
EGFP-positive cardiomyocytes were mounted on the slide glass. After
air drying, the slide glass (sample) was rinsed in 75 mmol/L KCl (Wako,
Tokyo,Japan) at room temperature for 60 minutes. The sample was fixed
in Carnoy!s solution (methanol 3 volumes, acetic acid 1 volume) 4 times
for 5 minutes each at 4ºC. After drying on a slide warmer at 65ºC for 10
minutes, the sample was rinsed in aging buffer (2xSCC, 0.1%NP-40) for
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