Dulbecco’s modified Eagle Medium (DMEM) high glucose, Neurobasal medium (NB), Hank’s balanced Salt Solution (HBSS), Earle’s balanced Salt Solution, l-glutamine, sodium pyruvate, penicillin/streptomycin, Diamidino-2-Phenylindole, Dilactate (DAPI) were purchased from Life Technologies (Carlsbad, CA, USA), N21-MAX medium supplement from R&D Systems (Minneapolis, MN, USA), normal goat and fetal bovine serum, forskolin, triiodothyronine (T3), vitamin B12, hydrocortisone, biotin, boric acid, apotransferrin, putrescine, progesterone, sodium selenite, poly-d-lysine, recombinant human insulin, bovine serum albumin and DMSO were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trace elements B and trypsin 0.05 %-EDTA were purchased from Mediatech, Inc. (Manassas, VA, USA). Human ceruloplasmin was purchased from EMD Millipore (Billerica, MA, USA). Recombinant human BDNF and CNTF were purchased from PeproTech (Rock Hill, NJ, USA). Laminin was obtained from Trevigen (Gaithersburg, MD, USA). DNase and papain were purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). Packard Viewplates 96-well were purchased from Perkin Elmer (Waltham, MA, USA). Additional file 13: Table S2 lists the primary antibodies and their dilutions used in this study.
Cell culture methods
All animal work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal protocols were approved by Institutional Animal Care and Use Committee (IACUC) at the Molecular Medicine Research Institute. Animals were either euthanized by CO2 asphyxiation or decapitation.
RGC-OPC culture methods
RGCs were prepared from P6-P7 Sprague–Dawley rat pups (Charles River, Wilmington, MA, USA), following the RGC immunopanning purification protocol as described in Watkins et al. . On DIV11 of RGC culture, cortical OPCs were purified from P7 Sprague–Dawley rat pups, following the OPC immunopanning purification protocol (as described in ). Six days following test compound addition (17 DIV), cells were fixed, immunostained and imaged as described below.
Embryonic cortical culture methods
The dissection of E18 rat (Charles River, Wilmington, MA, USA) cortex is similar to that described previously [31–33] with some modifications. Briefly whole cortices from three embryos were collected in a petri dish containing HBSS. After carefully removing the meninges, the tissue was divided into cortical hemispheres, dissected and the non-cortical structures were removed. Cortical tissue was then digested in 7 U/ml papain dissolved in HBSS with 500 U/ml DNase I, and incubated for 30 min at 35 °C. The enzymatic reaction was terminated with DMEM containing 10 % FBS. The tissue was allowed to settle, supernatant was removed and tissue was triturated with a flame-polished glass Pasteur pipette in DMEM/10 % FBS, 250 U/ml DNAse I until the tissue was completely dispersed. The dissociated cell suspension was centrifuged at 200×g for 5 min and supernatant replaced with plating medium (NB medium with 1× N21 supplement and 2 mM l-glutamine and 1 % penicillin–streptomycin). Viable cells were counted using trypan blue exclusion and typically exceeded 80 %. Isolated cells were seeded onto 96-well plates pre-coated with poly-d-lysine (10 μg/ml) and laminin (2 μg/ml) at a density of 20,000 cells/well (2 × 105 cell/cm3). Neurons were allowed to adhere, recover, mature and extend axons for 3 days. On the fourth day, the plating medium was diluted with an equivolume of myelination medium (MyM), as described in Watkins et al.  with minor modifications (see “Results”). The following day, two-thirds of the medium was replaced with fresh MyM and test compound. The day after establishing the primary culture was defined as day 1 in vitro (DIV1).
Acute oligodendrocyte differentiation assay
OPCs from P7 Sprague–Dawley rat pups were purified by immunopanning and cultured as described . OPCs were plated at 5000 cells/well into pDL-Laminin coated 96-well TC plate wells and centrifuged at 200×g to facilitate cell attachment, survival, and assure even distribution of OPCs. Plated OPCs were pre-incubated for 1–2 h at 37 °C in 10 % CO2 incubator, followed by addition of test compounds in quadruplicate. Controls were added in eight replicate wells, negative control = 0.1 % DMSO final concentration; positive control = 40 ng/ml T3. The day of OPC plating was considered DIV0. On DIV4, cells were fixed, immunostained, and imaged as described below. Minor modifications include blocking cells with 10 % normal goat serum (NGS)/0.4 % Triton X-100 and staining overnight at 4 °C with rat anti-MBP antibodies diluted in 10 % NGS/0.08 % Triton X-100. OL differentiation was quantified by IN Cell Developer Toolbox image analysis software which calculated the MBP staining intensity of two images per well. The extent of OL differentiation was defined by the total threshold-selected area of MBP staining x MBP fluorescence intensity in this area divided by the total number of OLs (identified by DAPI nuclear staining).
Immunofluorescence staining and imaging
At the experimental end point, medium was removed leaving 50 μl/well using an ELX405 microplate washer (BioTek, Winooski, VT, USA). Cells were then fixed for 14 min with paraformaldehyde solution to a final concentration of 4 %. Following fixation, plates were washed with 1 ml PBS leaving 50 μl/well using the microplate washer. Cells were then blocked in blocking buffer (10 % normal goat serum, 0.1 % Triton X-100), antibody buffer (150 mM NaCl, 50 mM Tris Base, 1 % BSA, 100 mM l-lysine, 0.004 % sodium azide, pH 7.4), and stained with mouse anti-rat MBP antibody and anti-rabbit Olig2 diluted in blocking buffer overnight at 4 °C. The cells were washed and incubated with secondary antibodies, and DAPI, 0.3 μM for 1 h at room temperature. After a final wash, 100 μl of PBS was added to each well and plates imaged. Images were captured with a Nikon Eclipse TE-2000-U microscope, Zyla cMOS megapixel camera (ANDOR Technology, Belfast, UK), fitted with an automated stage controlled by NIS Elements AR software 4.0 (Melville, NY, USA). An air 10X lens was used to capture four images per well with 16 bit resolution, 2560 × 2160 pixels. Images for each assay run were captured using identical camera settings. Images were exported as TIFF files for analysis and quantification.
TIFF files were analyzed using a custom algorithm created with IN Cell Investigator Developer Toolbox (GE Health Sciences, Piscataway, NJ, USA). For each well, four images were analyzed and the data from the duplicate well combined and averaged (total of eight images per test condition). The extent of OL differentiation was defined by the total threshold-selected area of MBP staining × MBP fluorescence intensity in this area divided by the total number of OLs (identified by Olig2 nuclear staining). We referred to this as the “MBP score” or “OL differentiation”. Earlier publications have characterized in vitro myelination as contiguous segments of MBP staining co-localizing with axons, representing the contact and ensheathment of axons with the myelin membrane generated by OLs. Hence, our assay defined myelination as the alignment of MBP staining assuming the shape change into long straight contiguous segments. Our fiber length algorithm delineated only what it identified as continuous straight lines from an image field, and then applied morphometric quantification of intensity of those selected lines (calculates the total pixel length within a single fibrous shape). This value was then divided by the total number of OLs to give the value referred to as “fiber score” or “myelination”. The quotient of the myelination score and the MBP score equals a value we referred to as “fiber score/MBP score”, reflecting myelination independent of the effects of differentiation; in other words, MBP staining specific to myelination. Numerical results from the analyzed images were later exported for analysis in Microsoft Excel (Redmond, WA, USA). Data was normalized by fitting parameters to positive (1 μM DAPT) and negative controls (0.1 % DMSO) and expressed as the % of DAPT.
Relative EC50 analysis
Half maximal effect concentrations (EC50) values were obtained by fitting the data to a sigmoidal dose–response curve-fitting function (Prism, GraphPad software, La Jolla, CA, USA). Serial dilutions of eight to ten different concentrations with four data points per concentration were used for curve fitting. Experiments were repeated at least two times.
All compounds in the NCC library were supplied in DMSO at 10 mM in 96-well plates. Hit compounds were purchased as powders and stocks dissolved in DMSO to 10 mM for in vitro studies (see Additional file 13: Table S2 for complete listing of compounds). N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), LY411,575, and BMS 708163 were from Selleckchem, MRK560 was purchased from Tocris.
For all experiments, assuming normal distribution, two-tailed t-tests were used to evaluate comparisons between two groups and ANOVA was used when more than two groups were compared. For the quantitative analysis of in vitro myelination and differentiation, a one-way ANOVA was conducted to compare the effect of each dose of compound with DMSO (Prism, GraphPad software, La Jolla, CA, USA). This was followed by post hoc comparisons using Bonferroni or Dunnett Multiple comparison tests. Where possible, data were represented as the mean (M) ± standard error of the mean (SEM) or standard deviation (SD) unless otherwise indicated in the figure legends.