Growth factor depleted matrigel and matrigel coated invasion chambers were obtained from BD Bioscience

al. Maxadilan Prevents Apoptosis in iPS Cells reported that both PACAP and maxadilan could prevent TNFa-mediated cell death in olfactory placodal cells and that PACAP protects the mouse olfactory epithelium cells against axotomy-induced apoptosis. Racz B et al. reported that PACAP effectively protects cochlear cells against oxidative stressinduced apoptotic cell death. Gasz B et al. showed that PACAP was able to attenuate oxidative stress-induced cardiomyocyte apoptosis. In 2004,Cazillis M et al. demonstrated that PAC1 is expressed and functional in mouse embryonic stem cells. Soon after, Hirose M et al. also identified that PAC1 is present in ES cells. However, little is known about the presence and effects of PAC1 in iPS cells. In this study, the expression or absence of PAC1 in iPS cells was investigated, and maxadilan was subsequently used to probe the anti-apoptotic effects mediated by PAC1 in iPS cells. This research attempted to understand if maxadilan could be an additive to facilitate large-scale culturing of iPS cells. intensities was performed by scanning the immunostaining band and analyzing the image with ImageJ 1.39 software. Viability of iPS cells after UVC irradiation One hundred microliters of the single-cell iPS cell suspension were seeded onto each well of a 96-well plate coated with Matrigel. iPS cells were cultured in mTeSR1 medium in 96well plates to produce colonies at 80%90% confluence. Ten microliters of maxadilan were added to each well, and the plates were incubated at 37uC for 1 h. Cells were washed with phosphate buffered solution and subsequently ” exposed to 50 J/m2, 75 J/m2 and 100 J/m2 ultraviolet C at 254 nm. Fresh culture medium and the appropriate concentrations ” of maxadilan were added, and cells incubated for 24 h. Control wells contained iPS cells cultured in mTeSR1 medium and were not irradiated with UVC. iPS cell viability was measured by WST-8 analysis using the Cell Counting Kit-8 . The samples were stained with 10 ml of the CCK-8 solution and incubated on the plate in a CO2 incubator for 3 h. Absorbance of the iPS cells at a wavelength of 450 nm was spectrophotometrically measured with a microplate reader equipped with the Magellan software. Materials and Methods Cell culture conditions and drug treatments The UMC human iPS cell line was used in all experiments. This iPS cell line was established from the umbilical cord matrix and amniotic membrane mesenchymal cells by transduction of retroviral factors, EMA-401 web including Oct4, SOX2, c-Myc, and Klf4. The cells were cultured under feeder-free culture conditions. Briefly, iPS cells were cultured in mTeSR1 medium on dishes coated with Matrigel. The cells were grown in 5% CO2 with 95% humidity. The cell medium was changed daily, and spontaneously differentiated colonies were removed when appropriate. iPS cells were passaged every six days with 0.05% trypsin-EDTA at 37uC for 35 min. When colonies near the edge of the plate began to dissociate from the bottom, the enzyme was removed, and colonies were washed with mTeSR1 medium. Cells were collected by gently pipetting and replated onto fresh Matrigel-coated dishes. The ROCK inhibitor Y-27632 was added to each well for the first day after each passage. Annexin V and propidium iodide assays iPS cells were cultured in mTeSR1 medium in 6-well plates to produce colonies at 80%90% confluence. The iPS cells were irradiated with UVC as described above. The UVC30 nM maxadilan iPS samples were treated with 30 nM of max