Supplementary
Materials
Materials purchased from Fisher Scientific, Pittsburgh, PA were: T-75 flasks and 0.2% Triton X-100. Products from Sigma-Aldrich, St. Louis, MO were: poly-L-ornithine (10 µg/mL), agarose (42°C gelation temp), bovine serum albumin (BSA) and lactate dehydrogenase (LDH) assay kit. Similarly from Invitrogen/Gibco, Carlsbad, CA: phosphate-buffered saline (PBS), N2 Supplement, L-glutamine and 0.05% Trypsin-EDTA. Laminin-1 from BD Biosciences (Bedford, MA) was used at a concentration of 5 µg/mL on the T-75 flasks and 10 µg/mL for electrical stimulation chambers. Other materials used are: Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12, 1:1; Omega Scientific, Tarzana, CA), basic fibroblast growth factor (human recombinant bFGF; Promega Corporation, Madison, WI), fetal bovine serum (FBS, Atlanta Biologicals Inc., Laurenceville, GA), and filtered water (18 MW, Millipore, Billerica, MA).
Method Details or Notes
Cleaning of Stimulation Apparatus
The machined polycarbonate pieces and glass slides were cleaned by boiling in diluted RBS-35 detergent concentrate (Pierce, Rockford, IL). They were then rinsed repeatedly with purified, filtered water (18 MW) and sterilized by exposure to UV light for 30 minutes. Each glass slide was aligned to the longest side of its corresponding machined polycarbonate part before adhering the two parts. After allowing the silicon adhesive Sylgardâ (Dow Chemical, Midland, Michigan) to dry for two days, 70% ethanol was flushed through the chambers and the apparatus was rinsed with distilled water.
Image-J Semiautomatic Quantitative Analysis
In an attempt to automate cell counting, a macro was programmed using ImageJ [1]. Images were processed in batches, and for each image the background fluorescence was subtracted, DAPI/anti-BrdU immunoreactive nuclei that were approximately 15 to 315 µm2 were encircled, and counted automatically. The procedure was semi-automatic, as user intervention was required to determine the total cell count by manually counting nuclei with low fluorescent intensity, clumped nuclei, and/or areas out of the specified range. For each experimental condition, all nuclei counted were summed to determine the number of cells examined. Likewise, BrdU-immunoreactive (IR) nuclei were semi-automatically counted using the macro created for ImageJ. The total DAPI stained nuclei divided by the total actual area photographed yielded the cell density (cells per mm2) for each treatment.
Microscopy
Cells were observed using light microscopy (Nikon Diaphot-TMD bright field/phase contrast microscope) during culture. Images were captured with a charge-coupled device camera (Megaplus; Model 1.4; Kodak Corp., San Diego, CA) connected to a frame grabber (Megagrabber; Perceptics, Knoxville, TN, in a Macintosh computer; Apple Computer, Cupertino, CA) using NIH Image 1.58VDM software (Wayne Rasband, National Institutes of Health, Bethesda, MD). For immunocytochemical labeling, a Nikon TE2000 (Nikon Corp., Melville, NY) inverted microscope equipped with standard epifluorescence illumination and a cooled digital camera (Cascade® 512B or Coolsnap® EZ; Photometrics, Tucson, AZ) controlled by MetaMorph software (Universal Imaging Corporation, West Chester, PA) was used. Time-lapse videos were captured with a cooled mono-chrome 12-bit digital camera (Retiga; QImaging, Surrey, Canada), mounted on a Leica DMIRE2 (Leica Microsystems, Wetzlar, Germany) controlled through OpenLab v. 3.5.1.
Cell Viability
Lactate dehydrogenase (LDH) assay kit was used to determine the viability of cells after one day of treatment. LDH content in the media of each experimental condition was determined colorimetrically. Media from electrical stimulation setups used to nourish cells during time-lapse recordings was not used for the LDH assay. Colorimetric readings were normalized between 0 and 1, corresponding to positive and negative controls, respectively. Positive controls consisted of killed cells using 0.2% triton-X-100 in media. Negative controls were NPCs in the apparatus that received no EF treatment. Cell density was determined along with immunocytochemical quantification (see below).
Quantitative Analysis of Immunocytochemistry
Images of NPCs in the chambers were captured where the EF was uniform (as indicated in Figure 1, A). Ten to twelve areas were analyzed per primary antibody. Two to three images were taken for each field analyzed, corresponding to fluorescent emissions of DAPI, CY3 and/or CY5. Nuclei and immunoreactive (IR) cells were counted semi-automatically using Image J [1]. The sum of IR cells was divided by the sum of nuclei (DAPI-labeled) for all images per treatment to determine the percentage of immunoreactive cells for each primary antibody. In other words, the percentage of NPCs that differentiated into neurons, oligodendrocytes and astrocytes after 6 DIV and those that were proliferating 14 h before the end of the experiment, were determined for each experimental condition. The background or non-specific immunoreactive intensity was determined from cells that were exposed to fluorescent secondary antibodies (CY3/CY5) only. Only immunoreactive cells with emission intensities above the non-specific intensity threshold were counted.
Video
The supplementary time-lapse video provided shows NPCs approximately 6 h after seeding. A 437 mV/mm EF is applied as indicated (at ~9 h) and then the polarity is reversed (at ~45 h) as indicated by the arrows and the stamp at the top left corner. The influence of the electrical field can clearly be seen since cells migrate toward the cathode (-) in the direction of the EF vector even after the EF vector is reversed.
Video Discussion
Based on morphology the direction of cellular migration differed in rare instances. We noticed that some flat astrocyte-like cells migrated in the direction opposite to the EF vector. In the supplementary video provided a morphologically flat cell can be observed emerging from the top left corner to the right of the time stamp at approximately 31 h; that cell’s migration is directed toward the anode (+) counter to the migration of the majority of cells even after the EF polarity was reversed. This is likely attributed to changes in properties once NPCs have differentiated or have become committed to a cell fate. For example the dielectric properties of NPC progeny were found to be distinct, allowing sub-population of cell types to be separated using various EF frequencies[2].
References
1. Abramoff MD, Magelhaes PJ, Ram SJ. Image Processing with ImageJ. Biophotonics International 2004;11(7):36-42.
2. Flanagan LA, Lu J, Wang L, et al. Unique dielectric properties distinguish stem cells and their differentiated progeny. Stem Cells 2008;26(3):656-65.