XB-IMG-133103
Xenbase Image ID: 133103
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Figure 2 Differentiated cells under the microculture condition. (A) Differentiated cell morphologies. Dissociated animal cap cells cultured
in different conditions were observed under phase-contrast. Representative morphologies are shown. a: Dissociated animal cap cells
cultured in control condition. The cells developed nerve fibers (arrowheads). Bar = 200 μm. b, c: Dissociated animal cap cells incubated
in CMFM with 40 ng/mL rhBMP-4. Bar = 100 μm. (b) An aggregate of ciliated cells. (c) A sheet of ciliated cells attached to the culture
dish. (B) Immunostaining of the differentiated cells. Cells were fixed after incubation for 36 h and examined for the presence of AP-20 or
E3 antigens. a, b: Cells with nerve fibers are stained with mAb AP-20, which is highly reactive with the microtubule-associated protein
2 (MAP2). This antibody stains throughout dendrites and somas. c, d: Ciliated cells stained with mAb E3, which selectively recognizes
differentiated epidermal cells. Nuclear staining was carried out with PI (b, d). Bar = 100 μm. (C) RT-PCR analysis of the differentiated
cells. Dissociated animal cap cells prepared from stage 8 were incubated in CMFM for 4 h. Then, the cells were cultured for 18 h in
NAM/2 before assessing the cell differentiation by RT-PCR. NCAM is a general neural marker: keratin for epidermis: Xbra for mesoderm:
and EF1-α for loading control. The animal cap cells that were treated with the culture medium derived from 1.5 ng BMP-4
mRNA- and H2O-injected oocytes expressed keratin (lane 1) and NCAM (lane 2), respectively. Intact animal caps expressed keratin (lane
3), while the whole embryos at stage 25, which is accounted for the processing time of 18 h from stage 8, exhibited all the marker transcripts
examined thus far (lane 4). The ‘–RT’ contained all reagents except reverse transcriptase and was used as a negative control (lane 5). Image published in: Nakajima Y et al. (2009) Copyright © 2009. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View |