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Cardiomyocytes isolation and culture Rat neonatal cardiac tissues were collected and kept in a head over head rotator at 4 C in trypsin overnight. Afterwards, the tissues were enzymatically digested with 550 U of Collagenase A, and filtered through 70 um cell straine into ABT-888 価格 the cold FCS solution. The cell sus pension was resuspended in DMEM, 10% FCS, 100 U mL penicillin, 100 mg mL streptomycin and 2 mM L glutamine. Fibroblasts were depleted through plastic adhesion, non adhered cells i. e. cardiomyocytes were re seeded at 20,000 cells cm2 in fibronectin coated flasks. Animal experiments, i. e. the use of neonatal rat heart for the isolation of cardiomyocytes was approved by the local committee for animal experiments of the Amsterdam University Medical Centre.

Animal experimentation Afatinib 溶解度 was approved by the local committee for care and use of laboratory animals and performed according to strict governmental and international guidelines. The investigation conformed to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. HL 1 murine cardiomyocytes were a kind gift of Dr. William C. Claycomb. Cells were main tained in fibronectin coated flasks in Claycomb expansion medium supplemented with 10% FBS, 0. 1 mM norepin ephrine, 100 U mL penicil lin, 100 mg mL streptomycin and 2mM L glutamine and kept semi confluent at all times. Experimental culture conditions Prior to co cultures of ADSC and rat neonatal car diomyocytes the cells were labeled with the CFDA SE and CM DiI respectively according to the manufacturer s instructions.

Co cultures of ADSC and HL 1 cardiomyocytes were done after lentivirus tagging AG-1478 分子量 with resp. lentiviruses encoding eGFP and dTomato. Briefly, on the day of transduction, cells were plated at 1× 106 cells well in serum free growth medium containing 5 ug ml polybrene . Following overnight incubation, medium was replaced with normal growth medium containing 10% FBS. The medium of HL 1 cells was changed once per 24h while ADSC medium was replenished three times a week. At five days post transduction, cells were FACS sorted based on expression of eGFP or dTOMATO to obtain pure cell population. To determine the influence of the ADSC density on cardiomyocyte proliferation, ADSC were treated with 10 ug ml mitomycin C for 3h, followed by extensive washing with PBS prior the co culture with rnCM and HL 1 cells.

The ADSC cell ratios plated in co culture conditions varied from 1,1 to 1,3 for rnCM, while keeping the rnCM at 20,000 cells cm2. The ADSC ratios in co cultures with HL 1 cells varied from 1,1 to 1,4, while keeping the HL 1 cells at 6,000 cells cm2. Simultaneously, cells were labeled with 1 uM BrdUrd bromodeoxyuridine for 6h at the end of the experiment. In order to study the effect of the post MI microenvironment on cardiomyocytes, rnCM and HL 1 cells and ADSC were cultured at ambient oxygen tension 21% O2 or at 2% O2,. At these oxygen tensions inflamma tion was mimicked by continuous treatment with TNF or IL 1B or none as a control for 24 and 48h respectively. ADSC conditioned medium was collected after pre treatment according to the experimental procedures for 24h.