Table 1.
Primer Sequences 5'-3'.
Fig 1.
MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP.
Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.
Fig 2.
Morphological differentiation of MED17.11.
A, Protocol for differentiation of MED17.11 cells. Bright-field images (B and C) of MED17.11, 3 days after the addition of differentiation medium. B, Low magnification images illustrate the high efficiency of morphological differentiation using the protocol shown in A. The small black arrow points to the rarer large diameter neurons. C, Higher power images showing the typical morphology of MED17.11 cells. The large black arrow points to an example of process branching. D, Frequency distribution of cell soma diameters (left) demonstrates the diversity of cell sizes following differentiation. E, Histogram showing change in soma diameter post differentiation (right) * = p<0.05, ** = p<0.001 (mean + S.E.M; Student’s unpaired two tailed t test). F, Immunofluorescence images of MED17.11, 3 days after the addition of differentiation medium. Both soma and processes of MED17.11 cells label with the neuronal marker, Tuj1, and the sensory neuron marker, Advillin. All scale bars are 50 μm.
Fig 3.
mRNA and Immunocytochemical profiles of MED17.11.
Left, mRNA analysis of proliferating and differentiated MED17.11 cells: “Undiff” refers to cells grown in proliferating conditions (see methods). “Diff” refers to cells grown in differentiating conditions for 7 days (day 9 of differentiation protocol in Fig 2, also see methods). A “+” indicates that the cell line was positive and a “-” indicates that the cell was negative for the corresponding marker. A”?” indicates that the marker was not tested in the given condition A. The “*” beside Runx1 indicates that this nociceptor marker was also tested after one day in differentiation conditions and was positive. LTM = low threshold mechanoreceptor, VGSC = voltage gated sodium channel. Right, immunocytochemical analysis of MED17.11 cells differentiated for 7 days. The cells showed immunoreactivity for the modality specific markers TrkA and TrkC (top) and the embryonic VGSC, Nav1.3 (bottom left). Live MED17.11 cells also strongly labelled with IB4 (bottom right). Scale bar is 50 μm.
Fig 4.
Differentiated MED17.11 responds to compounds that excite sensory neurons and inflammatory mediators.
A, representative traces from fura-2 AM calcium imaging showing responses to compounds that excite DRG neurons following 3–6 days of differentiation. Dotted Lines indicate the time when the stimulus was applied. B, left, percentage of proliferative and differentiated cells responding to each compound (Fisher’s Exact two tailed test), * = p<0.05, ** = p<0.001, ***p<0.0001. Right, response amplitudes to each compound (mean + S.E.M). C, Percentage of MED cells responding when grown in non-permissive conditions with growth factors alone (NGF 100 ng/ml and GDNF 20 ng/ml) for 3 days. D, representative traces from fura-2 AM calcium imaging showing responses to inflammatory mediators following 3–6 days of differentiation. Dotted Lines indicate the time when the stimulus was applied. E, left, percentage of cells responding to each compound; right, response amplitudes to each compound (mean + S.E.M).