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

More documents

Recommendations

Info

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 Suppl 10
Tsuji et. al. amniotic membrane-derived stem cell (2009/205260-R3) 30 minutes at 37ºC, and then dehydrated in 70%, 85%, 100% ethanol for 1 minute each at 4ºC. The sample was denatured in denaturation buffer (70% formamide, 2x SCC) for 5 minutes at 37ºC and dehydrated again in the same way as above. Human Y-FITC conjugated (Cambio, STARFISH, 1083-YF-02), human Alu (BIOGENEX, PR-100101), ratXbiotin conjugated (Cambio, STARFISH, CA1699-XB), and hybridization buffer (Cambio, STARFISH, HYB-1-10) for the negative control were applied onto the cells, covered with a coverglass and sealed with a paper bond. Hybridization was performed by Hybridizer (DakoCytomation, S245030) at 95ºC for 10 minutes, then 42 for overnight. On the following day, after removing the cover glass, the glass slide was rinsed in 50% formamide (Nakarai, Tokyo, Japan) at 2SSC, pH7.0 /HCl 4 times for 10min each at 45, 2SSC twice for 10min at 45, then at room temperature. Preblock was done in 1% block ace powder (Dainihonseiyaku, Tokyo, Japan)/ 2SSC for 20 min at room temperature, followed by blocking with Avidin/Biotin Blocking kit (Vector Laboratories, SP-2001) per manufacturer!s recommended protocol, biotinylated anti fluorescein antibody (Vector Laboratories, BA-0601) was applied for 30 min at room temperature. After rinsing in 4SSC three times at room temperature, Streptavidin-Cy2 (Jackson Immuno Research, 016-220- 084) and DAPI were applied for 30 min at room temperature. Finally, the glass slide was mounted with fluorescent mounting medium (Dako Cytomation, S3023) and inspected with a laser confocal microscope (FV1000, Olympus, Tokyo, Japan). 6. Teratoma formation assay After hAMCs transplantation, histological analysis was performed to observe teratoma formation 5 of EGFP positive cells. Samples were examined for 89 recipient!s hearts. 21.3 ± 2.0 days after transplantation, Suppl 11
Page 1 and 2: Tsuji et. al. amniotic membrane-der
Page 9: Tsuji et. al. amniotic membrane-der
Page 13: Tsuji et. al. amniotic membrane-der

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

Create successful ePaper yourself

Delete template?

Save as template?