Sts were induced to differentiate and fuse into myotubes by replacing the culture medium with DMEM and 2 horse serum. Undifferentiated NG108-15 cells were maintained in medium containing DMEM supplemented with 10 FBS, 100 mM hypoxanthine, 15 mM thymidine and 1 mM aminopterin. NG108-15 cells were induced to differentiate by adding 1 mM of dibutyryl cAMP (Sigma) to the culture medium. The differentiation of NG108-15 cells was monitored by scoring neurite-positive cells (neurite defined as processes .26 soma diameter). Normally, more than 90 of the cells were neuritepositive 2 days after induction with dibutyryl cAMP, as compared to less than 10 of neurite-positive cells in the undifferentiated culture. To test the neurotrophic effects of BMP4, differentiated NG108-15 cells were CP21 manufacturer treated with 50 mM glutamate (in DMEM supplemented with 10 mM glycine) and/or 10 ng/ml BMP4 for 12 hours. Afterwards, cells were returned to their original culture medium (conditioned medium), with or without BMP4 for 8 hours. Cell viability was then determined under a microscope (106 eyepiece and 106 objective lens) by trypan blue exclusion.OperationsAdult mice were anaesthetized with isoflurane gas (2? by volume, 0.4 L/min). The left hypoglossal or sciatic nerve was double ligated as previously described [22]. A midline incision was made in the ventral neck and the tendon of the digastric muscle sectioned to expose the hypoglossal nerve. The nerve was then ligated in two places, 1? mm apart, using fine surgical thread. The sciatic nerve was exposed by separating the anterior border of the biceps femoris from other structures and ligated as described for the hypoglossal nerve. Eighteen to 20 hours after ligation, the animals were killed in a CO2 chamber and their nerves dissected. In some experiments, the extensor digitorum longus (EDL), soleus, and tibialis anterior muscles were denervated by sectioning the sciatic nerve in one of the hind limbs, as previously described [44]. The mice were killed by cervical dislocation 7 days after the operation. The muscles were removed and either processed for RNA analysis or snap frozen in melting isopentane for immunohistochemical analysis.ImmunostainingThe whole-mount tissues, cryosections or culture cells were stained by immunohistochemistry and immunocytochemistry, as previously described [19,23]. Antibodies included anti-BMP4 (Santa Cruz Biotechnology), anti-BMPRII (R D System), antisynaptophysin (Abcam), anti-neurofilament 200 (Sigma), antiS100 beta (Abcam). Immunoreactivity was visualized using fluorescent Alexa 
Fluor 350, Alexa Fluor 488 and BODIPY-fl conjugated secondary antibodies (Invitrogen) or using 3-amino-9ethylcarbamide (AEC) (Sigma) as the chromogen. NMJs were labeled with PS-1145 rhodamine-conjugated-a-bungarotoxin (BTX, Mo-AcknowledgmentsWe thank the staff of the cell imaging core at the First Core Labs, National Taiwan University College of Medicine, for technical assistance.Author ContributionsConceived and designed the experiments: HJC DML CWH ISM HDW PYW. Performed the experiments: HJC DML CWH PYW. Analyzed the data: HJC PYW. Contributed reagents/materials/analysis tools: ISM HDW. Wrote the paper: PYW.
Mast cells are derived from CD34+ hematopoietic progenitor cells, and play an important role in both innate and acquired immunity [1]. Mature mast cells express the stem cell factor (SCF) receptor, c-Kit (CD117), and the high affinity IgE receptor, FceRI [2]. Upon the aggregation of FceRI, induced by polyvale.Sts were induced to differentiate and fuse into myotubes by replacing the culture medium with DMEM and 2 horse serum. Undifferentiated NG108-15 cells were maintained in medium containing DMEM supplemented with 10 FBS, 100 mM hypoxanthine, 15 mM thymidine and 1 mM aminopterin. NG108-15 cells were induced to differentiate by adding 1 mM of dibutyryl cAMP (Sigma) to the culture medium. The differentiation of NG108-15 cells was monitored by scoring neurite-positive cells (neurite defined as processes .26 soma diameter). Normally, more than 90 of the cells were neuritepositive 2 days after induction with dibutyryl cAMP, as compared to less than 10 of neurite-positive cells in the undifferentiated culture. To test the neurotrophic effects of BMP4, differentiated NG108-15 cells were treated with 50 mM glutamate (in DMEM supplemented with 10 mM glycine) and/or 10 ng/ml BMP4 for 12 hours. Afterwards, cells were returned to their original culture medium (conditioned medium), with or without BMP4 for 8 hours. Cell viability was then determined under a microscope (106 eyepiece and 106 objective lens) by trypan blue exclusion.OperationsAdult mice were anaesthetized with isoflurane gas (2? by volume, 0.4 L/min). The left hypoglossal or sciatic nerve was double ligated as previously described [22]. A midline incision was made in the ventral neck and the tendon of the digastric muscle sectioned to expose the hypoglossal nerve. The nerve was then ligated in two places, 1? mm apart, using fine surgical thread. The sciatic nerve was exposed by separating the anterior border of the biceps femoris from other structures and ligated as described for the hypoglossal nerve. Eighteen to 20 hours after ligation, the animals were killed in a CO2 chamber and their nerves dissected. In some experiments, the extensor digitorum longus (EDL), soleus, and tibialis anterior muscles were denervated by sectioning the sciatic nerve in one of the hind limbs, as previously described [44]. The mice were killed by cervical dislocation 7 days after the operation. The muscles were removed and either processed for RNA analysis or snap frozen in melting isopentane for immunohistochemical analysis.ImmunostainingThe whole-mount tissues, cryosections or culture cells were stained by immunohistochemistry and immunocytochemistry, as previously described [19,23]. Antibodies included anti-BMP4 (Santa Cruz Biotechnology), anti-BMPRII (R D System), antisynaptophysin (Abcam), anti-neurofilament 200 (Sigma), antiS100 beta (Abcam). Immunoreactivity was visualized 
using fluorescent Alexa Fluor 350, Alexa Fluor 488 and BODIPY-fl conjugated secondary antibodies (Invitrogen) or using 3-amino-9ethylcarbamide (AEC) (Sigma) as the chromogen. NMJs were labeled with rhodamine-conjugated-a-bungarotoxin (BTX, Mo-AcknowledgmentsWe thank the staff of the cell imaging core at the First Core Labs, National Taiwan University College of Medicine, for technical assistance.Author ContributionsConceived and designed the experiments: HJC DML CWH ISM HDW PYW. Performed the experiments: HJC DML CWH PYW. Analyzed the data: HJC PYW. Contributed reagents/materials/analysis tools: ISM HDW. Wrote the paper: PYW.
Mast cells are derived from CD34+ hematopoietic progenitor cells, and play an important role in both innate and acquired immunity [1]. Mature mast cells express the stem cell factor (SCF) receptor, c-Kit (CD117), and the high affinity IgE receptor, FceRI [2]. Upon the aggregation of FceRI, induced by polyvale.