These results confirm that cells differentiated under reduced FSK and corresponding growth factors are capable of retaining a similar level of neurite length reported previously.1,2 However, the trade-off in acquiring longer term cultures is the loss of additional growth factor-mediated neurite outgrowth. Open in a separate window Figure 2. NGF or GDNF do not contribute significantly to the morphological differentiation of 50B11 cells. NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome N106 expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells. (DIV) post-FSK and growth factor treatments (50?ng/ml recombinant mNGF, Peprotech), GDNF (50?ng/ml recombinant hGDNF, Peprotech), and all subsequent protocols were designed to be completed within this time frame. Cells were then N106 used for either immunohistochemistry, lysed for Western blotting or qRT-PCR. Neurite outgrowth assays Following culture and treatments described above, 50B11 cells were photographed live on an Olympus (IX71) microscope. Measurements of neurite length (minimum 100 cells) were performed on 16-bit TIFF format images using the NeuronJ plugin written for Fiji (Image J, NIH). Single neurons with minimal or no overlapping of neurite arbours with adjacent N106 cells were analysed using NIH ImageJ software with the NeuronJ Plugin. Distances from soma perimeter to N106 neurite tips were measured by tracing arbours and expressed as the summed length of neurite outgrowth and as length of the longest axon. Phase annuli counts of the cell soma within each image were performed to determine % of differentiated cell in total populations. All data are presented as??standard error of mean (SEM), and treatment effects were compared by MannCWhitney rank sum tests. Western blot Treated 50B11 cells were lysed using chilled lysis buffer containing 10?mM Tris-HCl, pH 8.0, 150?mM NaCl, 2?mM EDTA, 1% NP-40, 1% Triton X-100, 10% glycerol, 1?mM phenylmethanesulfonyl fluoride, 1?mM sodium orthovanadate, 1?M batimastat (BB-94), and 1% Roche protease inhibitor cocktail (2). For Western blots, lysates were solubilized in an equal volume of 2??SDS sample buffer containing 4% SDS, 2% glycine, 0.015% bromophenol blue, 20% glycerol, and 10% -mercaptoethanol in 100?mM Tris-HCl buffer, pH N106 6.8. Protein quantification for all samples Rabbit Polyclonal to ACOT1 was performed with the BCA Protein Assay Kit (Pierce – Thermo Scientific). Cell lysates were electrophoresed through 4C12% Bis-Tris buffered SDS gels (Life Sciences). Proteins were transferred onto PVDF or nitrocellulose membrane at 100?V for 1?h. The membranes were blocked in 4% skim milk powder for transmembrane protein detection, or 3% bovine serum albumin (BSA; Sigma) for phosphorylated proteins, in 0.1% Tween-20, and 0.02% NaN3 in TBS, pH 8.0, for 1?h at room temperature and incubated overnight with primary antibodies at 4C. The following antibodies were used for western blotting: rabbit anti-p75NTR #9992 (1:5000, M. Chao), rabbit anti-TrkA (1:500; Biosensis), anti-TrpV1 antibodies (1:500; Biosensis), c-Ret (1:2000; Cell Signalling), anti-P2X3 (1:500; Cell Signalling), GFR1 (1:200, goat antiserum, R&D Systems) and mouse anti–III tubulin (1:2000; Promega). Membranes were then washed three times in TBS-Tween 20 (TBST), pH 8.0, for 10?min and incubated for 1?h with donkey anti-rabbit 680 or donkey anti-mouse 800 secondary (1:50000; Invitrogen) in TBS at room temperature and then washed three times in TBS for 10?min. For stripping and re-probing Western blot membranes were treated with 10?ml of Restore? PLUS Western blot stripping buffer (Thermo-Fisher) for 30?min at room temperature on an orbital shaker, washed three times with TBST and blocked with either 5% skim milk powder or 3% BSA in PBST for 1?h and then incubated with appropriate primary and secondary antibodies as described above. Western blots for individual markers were performed.