We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Rodent models of genetic diseases have proven useful in establishing the physiological functions of normal proteins and nucleic acids, as well as the pathophysiological consequences of defects in these. Examples include mice deficient for proteins involved in key cellular functions, as well as mouse models of disorders such as Alzheimer's disease. However, certain genetic manipulations can lead to neonatal lethality shortly or a few days after birth. In these cases, primary cell cultures are an important tool because live cells can be obtained from the embryonic or neonatal pups before death, they can be maintained for at least a few weeks in vitro, and during this time early neuronal development can be followed by biochemical, functional and morphological experiments. For the primary cultures, it can be beneficial to plate the neurons at low density; this makes it possible to visualize the individual somata, dendrites, axonal shafts and nerve terminals at high spatial resolution. However, the survival and differentiation of neurons at low density typically requires that they are plated on a glial feeder layer, co-cultured with glial cells in the absence of physical contact with them, or cultured in medium conditioned by glia 1.
The establishment of low-density neuronal cultures on glial feeder layers can be dependent on fast and reliable genotyping beforehand – within a few hr in contrast to a few days. Speed is especially important when the neuronal genotype needs to be matched to that of a glial feeder layer prepared beforehand. As a more practical example, it may be necessary to decide which pups of which genotype to use in generating cultures, to optimize the efficiency of an experiment.
Here we demonstrate the working protocol that has been used for fast, simplified and reliable mouse genotyping in previous publications 2-6. Mouse tails and a commercially available kit are used. This protocol includes single-step extraction of nucleic acids from the tissue, and requires neither a nucleic-acid purification step nor use of a termination buffer ('stop solution'). The reliability of this genotyping method is illustrated by presenting the results of a series of tests when differences are introduced with respect to the starting amount of the specimens, the age of the animals and the length of the PCR amplicons. This kit offers the advantages of automated extraction and reliability.
For the sake of being comprehensive, the use of tattooing for long-term identification of the genotyped mice is also demonstrated. Tattooing is achieved by applying tattooing ink to the dermis of the skin (under the epidermis) 7. A procedure is described for tattooing the paw pads of newborn or 1 day old mice, although tattoos can be applied to other parts of the body, such as tails and toes, and to animals of all ages. In addition, procedures will be demonstrated for plating and culturing mouse neurons at a low density, based on optimized preparation of different types of glial feeder layers 2,8.
We use a genetic mouse model of the inherited neurological disorder DYT1 dystonia – an autosomal-dominant movement disorder caused by a mutation in the gene TOR1A (c.904_906delGAG/c.907_909delGAG; p.Glu302del/p.Glu303del) 9. The encoded protein, torsinA, belongs to the “ATPases associated with diverse cellular activities” (AAA+) family of proteins, whose members generally perform chaperone-like functions, assisting in: protein unfolding, protein-complex disassembly, membrane trafficking, and vesicle fusion 10-13. The mutation results in an in-frame deletion of a codon for glutamic acid, and can lead to manifestation of 'early-onset generalized isolated dystonia' 14,15. However, the pathophysiological mechanisms responsible for this disorder remain poorly understood. In a knock-in mouse model, the mutant allele is Tor1atm2Wtd, mentioned hereafter as Tor1aΔE. Heterozygous ΔE-torsinA knock-in mice are viable and genetically mimic human patients with DYT1 dystonia, whereas homozygous knock-in mice die after birth 16,17, with the latency to postnatal death affected by genetic background 18. The early death of homozygous knock-in mice necessitates that both the genotyping of animals and the establishment of neuronal cultures are completed rapidly. As another example of genotyping, Tfap2a (transcription factor AP-2α, activating enhancer binding protein 2α) will be used. The protein encoded by this gene is important in regulating multiple cellular processes, such as proliferation, differentiation, survival and apoptosis 19.
NOTE: All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the University of Iowa.
1. Long-term Identification of Mice Using Tattooing the Paw Pads
2. Genotyping Newborn Mice Using a Fast PCR Genotyping Kit
3. Primary Culture of Mouse Brain Neurons on Glial Feeder Layer
NOTE: The procedures for brain dissection and cell dissociation (3.1) are common to all the subsequent procedures. The procedures for mouse glial cultures (3.2), rat glial cultures (3.3), and mouse neuronal cultures (3.4) are described separately afterwards.
As an example of the application of this protocol, representative results are shown for labeling mice by tattooing, reliable genotyping under various experimental conditions, and establishing primary neuronal cultures on glial feeder layers.
Tattooing
Newborn pups were labeled on the paw pads using a tattooing system ('Newborn' in Figure 1). The labels remained clearly visible at 3 weeks ('3-week-old') and 32 weeks of age ('32-week-old'). The individual mice can be uniquely identified by a combination of tattoos on the four paws (numbers 1-16) and by the information about the animal cage, or more complex numbering schemes (not shown).
Genotyping
One representative example of genotyping is shown (Figure 2). The Tor1a gene of wild-type (Tor1a+/+), heterozygous (Tor1a+/ΔE) and homozygous (Tor1aΔE/ΔE) ΔE-torsinA knock-in mice was amplified from the genomic DNA isolated from tail clips of newborn pups. Genotyping in this specific example is based on the presence of a single 34-base-pair loxP site in the mutant Tor1a allele after successful Cre recombination and deletion of a neomycin resistance cassette 16,18.
Genomic DNA was extracted with minimal hands-on time, and many samples were processed simultaneously. The extraction volumes were kept constant, and therefore no adjustment of the volume was necessary to accommodate the changes in conditions tested. The reliability of genotyping was tested under four conditions, as detailed below.
First, the effect of differences in the amount of starting tissue was examined (Figure 3). Tails of different lengths were prepared from the heterozygous ΔE-torsinA knock-in mice (Tor1a+/ΔE) at weaning age (~3 weeks). Tail lengths of 2 to 5 mm, the range typically recommended in genotyping protocols, gave the same genotyping result of high quality. The two bands correspond to the wild-type and mutated alleles (Tor1a+ and Tor1aΔE, respectively).
Second, the effects of variability in animal age were examined (Figure 4). Tails were obtained from newborn, 3-week-old and ~24-week-old heterozygous ΔE-torsinA knock-in mice (Tor1a+/ΔE). Homozygotes were not used because they die several days after birth 16-18. The two expected bands for wild-type and mutated Tor1a genes were visible regardless of animal age (Figure 4A). The general applicability of this result was tested using a second gene, Tfap2a19 in wild-type mice (Tfap2a+/+) (Figure 4B).
Third, the effects of differences in length of the PCR amplicons were tested (Figure 5). For this purpose, different primer pairs were synthesized for a given gene, such that the amplified DNAs are of different base pair lengths. The Tfap2a gene was consistently detected with different amplicon lengths.
Fourth, two DNA extraction methods were compared (Figure 6). In one method, PCR strip tubes and the PCR thermal cycler were used (described in step 2). This is an automated, parallel multi-tube extraction method ('Auto' in Figure 6). Because multiple tubes can be handled simultaneously in strip format, and a PCR machine is used with a program to operate in four temperature steps, there is no need to transfer the tubes, manually change the temperature during the extraction or use multiple pieces of equipment. Thus it is easy to process many specimens. This was compared with the second method based on manual, single-tube extraction ('Manual' in Figure 6). This uses individual PCR tubes and separate non-PCR machines (heat blocks and water baths) to control temperature during DNA extraction. In this case, multiple, single tubes were moved to a new temperature after each step, requiring multiple temperature-controlling apparatuses. In both cases, the Tor1a gene was analyzed in wild-type, heterozygous and homozygous ΔE-torsinA knock-in mice (Figure 6A), and the Tfap2a gene was analyzed in wild-type mice (Figure 6B). The two extraction protocols yielded equivalent genotyping results.
In summary, the results presented in Figures 3 a 6 demonstrate that the genotyping method is robust, achieving reliable and reproducible results in spite of variations in tissue amount, animal age, amplicon length, and the extraction protocol used.
Neuronal cultures
For culturing mouse neurons on the mouse glial feeder layer, the order of the procedures is: (tattooing newborn mice → genotyping for glial culture →) glial culture → tattooing newborn mice → genotyping for neuronal culture → neuronal culture. For cultures established on the rat glial feeder layer, the procedures in parentheses are skipped.
We examined the supportive effect of the pre-seeded glial feeder layer on neuronal survival and growth. Neurons were obtained from the CA3-CA1 region of hippocampus, the motor region of cerebral cortex, and the striatum of newborn wild-type mice. They were plated at low density on rat glial feeder layer that had been obtained from the CA3-CA1 region of hippocampus and seeded prior to neuronal plating, according to the scheme illustrated in Figure 7 (simplified summary of the procedures). Low-density cultures are optimal for the imaging of individual dendrites, somata 4,6, nerve terminals 2,3,5,8 and axonal shafts 5 at high spatial resolution. The hippocampal cells (containing both neurons and glial cells) were plated on coated glass coverslips, either with (Figure 8A) or without a pre-established glial feeder layer (Figure 8B, C) and observed with phase-contrast optics. In the presence of a nearly confluent glial feeder layer, the neurons (in each panel, an arrowhead indicates one representative neuron) were relatively dispersed 3 and 7 days after plating (days in vitro, DIV) (Figure 8A). They also showed signs of good health, such as a clear margin of neuronal somata, extended dendrites, a lack of clustered somata, and a lack of bundled neurites (high-magnification images in insets). At 14 DIV, these neurons formed a dense network characterized by long neurites (dendrites and axons). Note that glial proliferation was inhibited before neurons were plated, by adding growth medium containing the mitotic inhibitor AraC (step 3.3.15).
In contrast, in the absence of the glial feeder layer at the time of plating hippocampal neurons, the growth of the cultured hippocampal neurons was impaired, even in the absence of AraC. At 3 DIV, glial cells (included in the newly plated hippocampal cells) have not formed a confluent sheet (asterisk in top panel of Figure 8B). The cultures showed several signs that are not appropriate for high-resolution imaging studies, such as wide areas without underlying glial cells (asterisk), the presence of clustered somata and the presence of bundled neurites in some areas (data not shown). By 7 DIV, glial cells formed a confluent sheet (middle panel of Figure 8C). However, neurons were fewer in number than when the neurons were plated on a pre-established glial feeder layer. The surviving neurons also lacked long, network-forming neurites (inset). The glial cells were more heterogeneous in curvature and thickness than those in the feeder layer culture, thus appearing phase-bright. In order to test the effects of suppressing glial proliferation on neurons, we applied AraC-containing growth medium. When AraC was added on 3 or 7 DIV and the cells were cultured until 14 DIV and observed on this day (Figure 8B and C, bottom panels), the glial cells covered most of the coverslip surface. However, the neurons were still few in number, especially when AraC was applied at 7 DIV. The surviving neurons had only short processes and were not extensively connected. Thus, the late addition of AraC allowed a uniform glial layer to form but did not promote neuronal viability or neurite extension; rather the uncontrolled glial growth had deleterious effects on neurons.
These data show that the glial feeder layer is critical for the survival and growth of neurons plated at low density, and that the glial layer must be present at the time of neuronal plating rather than later during neuronal culture. Similar results were obtained using cultures of cerebral cortical (Figure 9) and striatal neurons (Figure 10).
The qualitative observation about neuronal survival was confirmed by quantitative analysis. Specifically, the number of surviving neurons at 14 DIV was counted, based on phase-contrast images of the cultures (Figure 11). The number was greatest for neurons cultured on a glial feeder layer (i.e., plated on the glial feeder layer). The number was lower in the case of neurons cultured in the absence of a glial feeder layer. The number was reduced even further when the cells were treated with AraC at a later time. These differences were statistically significant, and were noted in cultures of neurons obtained from all three brain regions.
The types of surviving cells were identified at 14 DIV in the mouse hippocampal cultures plated on the rat hippocampal feeder layer. Double-immunocytochemistry was performed using antibodies against the neuronal marker, microtubule-associated protein 2 (MAP2), and the astrocytic glial cell marker, glial fibrillary acidic protein (GFAP). The cells with extended processes were positive for MAP2, whereas the cells in the underlying glial feeder layer were positive for GFAP (two representative image fields, Figure 12A). This staining was not an artifact of a specific combination of primary and secondary antibodies, because the same pattern was obtained when a different set of secondary antibodies was used (Figure 12B).
In contrast, when the same staining was performed on the cultures consisting of only a glial feeder layer, i.e., in the absence of added mouse cells (two representative image fields, Figure 13A), no cells were positive for MAP2. The cells of the feeder layer were consistently positive for GFAP. For a negative control, both primary antibodies were omitted from the immunocytochemical procedure (Figure 13B). No staining was detected in these samples, although cells were present, as evident from the transmitted light optics (differential interference contrast, DIC) and nuclear staining with Hoechst dye. Thus, non-specific staining in the MAP2 and GFAP channels was very weak.
These immunocytochemical data were also analyzed quantitatively (Figure 14). In each 8-bit image, intensity was measured and plotted along a line. Overlaid plots reveal MAP2 staining when mouse cells (samples containing neurons) were cultured on a glial feeder layer (Neuron +, glia +, primary antibody (1° Ab) +), whereas such staining was absent in cultures of glial feeder cells alone (Neuron −, glia +, 1° Ab +). In a negative control without primary antibodies, staining was negligible in cultures of glial feeder cells alone (Neuron −, glia +, 1° Ab −). GFAP staining was apparent in the glial feeder layer, regardless of whether mouse cells were plated (Neuron +, glia +, 1° Ab +) or not (Neuron −, glia +, 1° Ab +). Again, staining in the negative control was negligible (Neuron −, glia +, 1° Ab −). These results show that the rat glial feeder layer was composed mostly of astrocytes, and that the neurons were present only when mouse cells were added. They also indicate that the neurons present in these cultures originated solely from mouse.
The Results Section of the online video also shows the DIC and MAP2 images of cultured neurons plated on a glial feeder layer, both prepared from the CA3-CA1 hippocampal region of wild-type mice.
For detailed control of cell culture, it is recommended to measure the density of live cells, both for assessing the general quality of the brain dissection and cellular dissociation steps, and for plating the cells at the pre-determined density. Listed below are four sets of typical density measurements. Note, however, that these numbers can vary depending on culture conditions and reagents. For example, even different lots of serum from the same vendor can affect the results. Thus, these numbers should be considered a general indicator, rather than an absolute guideline.
For cellular dissociation (step 3.1.13), typical values for the live-cell density obtained from one pup are: ~2 x 105 cells/ml (mouse motor cortex and mouse CA3-CA1 hippocampus), ~5 x 105 cells/ml (mouse cerebral cortex and rat CA3-CA1 hippocampus), and ~1 x 106 cells/ml (mouse striatum). In all these cases, the fractions of live cells were >90% (viability, defined as the ratio of the number of live cells to that of the total number of cells). The motor cortex is loosely defined as the region of the cerebral cortex that lies immediately dorsal to the striatum 20. For hippocampal cultures, the CA3 and CA1 regions of the hippocampus proper are preferred. The whole hippocampus additionally includes the dentate gyrus, the inclusion of which leads to formation of large nerve terminals of granule cells and introduces heterogeneity in synaptic properties 21. The striatal culture includes the caudate-putamen and globus pallidus, but does not include the nucleus accumbens, or medial or lateral septal nuclei in the nearby structures.
For mouse glial culture (step 3.2.11), the plating density is ~10,000 glial cells on each 12-mm round coverslip, using ~50 μl of cell suspension at a density of ~2 x 105 cells/ml. Usually the density measured in the supernatant is relatively constant and does not require adjustment. To convert the density over different areas, the useful information is that the coverslip has a diameter of 1.20 cm and an area of 1.13 cm2. Thus, ~10,000 cells / coverslip = ~8,800 cells / cm2 on a coverslip.
For rat glial culture (step 3.3.12), the plating density is ~1,000 glial cells on each 12-mm round coverslip, using ~100 µl of cell suspension at a density of ~1×104 cells/ml. Thus, 1,000 cells/coverslip = ~900 cells/cm2 on a coverslip.
For low-density mouse neuronal culture (step 3.4.4), the plating density ranges between 2,000 and 48,000 cells per well of a 24-well plate. Typical values when plating neurons on rat glial cells are: 10,000-24,000 cells/well for cerebral cortical neurons, 12,000-24,000 cells/well for CA3-CA1 hippocampal neurons and 24,000-48,000 cells/well for striatal neurons. When plating on mouse glial cells, the number is reduced, e.g., 2,000 cells/well for CA3-CA1 hippocampal neurons. As a side note, for plating rat CA3-CA1 hippocampal neurons on a rat glial feeder layer, the plating density is 1,000-6,000 cells/well. For converting the density in a well to the density on a coverslip, the useful information is that the internal well diameter at the bottom is 1.56 cm and its area is 1.91 cm2. Thus, for example, 12,000 cells/well = 7,100 cells / coverslip = 6,300 cells/cm2 on a coverslip. These densities were chosen with the aim of culturing relatively sparse neurons for high-resolution cellular imaging. Researchers should choose their numbers that best suit their experimental aims.
Figure 1. Tattooed paw pads of mice. Newborn mice were labeled by tattooing the paw pads. They were photographed immediately after tattooing (newborn), at 3 weeks of age (3-week-old) and at 32 weeks of age (32-week-old). The labels remained easily visible throughout adulthood. Images were taken from different animals. They are not shown at the same scale. Please click here to view a larger version of this figure.
Figure 2. Genotyping of wild-type (Tor1a+/+), heterozygous (Tor1a+/ΔE) and homozygous (Tor1aΔE/ΔE) ΔE-torsinA knock-in mice. The Tor1a gene alleles were analyzed in wild-type and mutant forms, using DNA extracted from the tails of newborn pups. Tail tips were ~4 mm in length. DNA was stained using SYBR Safe DNA Gel Stain. See the Table of Materials/Equipment for the information about the primers and thermal cycling program for Tor1a gene. Please click here to view a larger version of this figure.
Figure 3. Detection of genomic DNA in the context of variation in the amount of starting material. Tail tips of different lengths were used. Tested animals were 3-week-old, heterozygous ΔE-torsinA knock-in mice (Tor1a+/ΔE). Lanes represent the tail lengths of 2, 3, 4 and 5 mm, in duplicate (from left). The tail tips were obtained from different mice. Molecular-weight size markers in the leftmost lane represent DNA lengths in steps of 100 base pairs. Please click here to view a larger version of this figure.
Figure 4. Detection of genomic DNA in the context of variation in animal age. (A) Tor1a gene. Lanes represent tail tips obtained from heterozygous ΔE-torsinA knock-in mice (Tor1a+/ΔE) at the following stages: newborn, 3-week-old, and ~24-week-old (in duplicates from left). (B) Tfap2a gene. Lanes represent tail tips obtained from wild-type (Tfap2a+/+) mice at the following stages: newborn, 3-week-old, and ~24-week-old (in duplicates from left). Both genes were amplified from tails ~4 mm in length. The expected PCR fragment is 498 bp. The PCR program was the same as that used for Tor1a gene. Please click here to view a larger version of this figure.
Figure 5. Detection of genomic DNA in the context of variation in length of the PCR amplicon. The Tfap2a gene was analyzed with PCR amplicon lengths of 498 bp (left lanes), 983 bp (middle lanes) and 1990 bp (right lanes) in duplicates. The gene was amplified from tails of 3-week-old mice. Molecular-weight size markers in the leftmost and rightmost lanes of the gel represent DNA lengths in steps of 100 and 200 base pairs, respectively. See the Table of Materials/Equipment for information about the primers for different amplicons. Please click here to view a larger version of this figure.
Figure 6. Detection of genomic DNA in the context of variation in DNA extraction method. Outcomes for the manual, single-tube extraction (Manual) and the automated, parallel multi-tube extraction methods (Auto) are compared. The latter method was used throughout this report. Genes were amplified from tails ~4 mm in length, collected from newborns. (A) Tor1a gene in the newborn pups of ΔE-torsinA knock-in mice. Lanes represent DNAs extracted from wild-type (manual and automated), heterozygous (manual and automated), and homozygous mice (manual and automated) (from left). (B) Tfap2a gene in 3-week-old wild-type mice. The expected PCR fragment is 498 bp. Please click here to view a larger version of this figure.
Figure 7. Simplified, schematic illustration of procedures for plating mouse neurons on mouse (A) and rat (B) glial feeder layers. The numbers of days (days in vitro) refer to the cumulative days after the glial cells are first plated in culture flasks. They serve only as a rough estimate. For practical details, see the Procedures section. Please click here to view a larger version of this figure.
Figure 8. Supportive effect of glial feeder layer on growth of hippocampal neurons in a low-density culture. Neurons were obtained from the CA3-CA1 region of the hippocampus of newborn, wild-type mice. (A) Mouse hippocampal cells were plated on coated glass coverslips, which had been pre-seeded with a rat glial feeder layer obtained from the CA3-CA1 region of hippocampus. Neurons were dispersed evenly in a healthy manner. Cultures were observed at 3, 7 and 14 DIV. (B, C) Mouse hippocampal cells were plated on coated glass coverslips, which had no pre-established feeder layer. Cultures were observed at 3 DIV (top panel in B) and 7 DIV (middle panel in C) without AraC treatment. In other sets of cultures, the cells were treated with AraC at 3 DIV (bottom panel in B) and 7 DIV (bottom panel in C), and observed at 14 DIV. All images represent the sister cultures obtained from the same pup, whose neurons were plated on the same day. See text for details. For each panel, an example of a neuron is indicated by a white arrowhead and magnified in an inset. Neurons have cell bodies whose perimeter appears bright when viewed by phase-contrast optics, and they also have thick processes. Asterisks indicate areas without glial cells. The cultures were imaged live on an inverted microscope using phase-contrast optics with 20X objective lens (numerical aperture of 0.45) without an intermediate lens (i.e., at 1x). Please click here to view a larger version of this figure.
Figure 9. Supportive effect of glial feeder layer on growth of cerebral cortical neurons in a low-density culture. Similar results as in Figure 8, but with neurons obtained from the motor region of cerebral cortex. The conditions under labels A-C correspond to those in Figure 8. Please click here to view a larger version of this figure.
Figure 10. Supportive effect of glial feeder layer on growth of striatal neurons in a low-density culture. Similar results as in Figures 8 and 9, but with neurons obtained from the striatum. The conditions under labels A-C correspond to those in Figure 8. Please click here to view a larger version of this figure.
Figure 11. Supportive effect of glial feeder layer on neuronal survival. The number of surviving neurons was counted in the experiments shown in Figure 8, using images acquired by phase-contrast microscopy. Images for 14 DIV are shown here again, but with arrowheads pointing to examples of counted neurons. The bar graph shows the number of neurons per image field (449.0 μm x 335.5 μm) (mean ± standard error of the mean, n = 7-24 fields for each culture condition). Asterisks indicate statistically significant differences of 'Glial feeder layer (-), AraC on 3 DIV' and 'Glial feeder layer (-), AraC on 7 DIV' from 'Glial feeder layer (+)' (* p <0.05, ** p <0.01, unpaired Student's t-test). The numbers above the bars indicate the neuronal density measured in montages of multiple image fields. Images were acquired using a 20X objective lens. To avoid acquisition bias, all images were acquired along a full vertical strip, from the top to the bottom of a coverslip and passing through the approximate center of the coverslip. Individual images had some overlap (e.g., 1/6 to 1/4 of an image field at the top and bottom). Two methods were used for the analysis. In one, neurons were counted in alternating images to ensure that individual neurons were not counted more than once. The resulting numbers were used to generate the bar graph. In the second method, a single montage was created from the individual images. They were stitched using the Microsoft Image Composite Editor or manually using Photoshop. The montages also excluded multiple counts of the same neurons. The resulting numbers are listed above the bars. In both methods, wide areas at the coverslip periphery that did not contain any cells were excluded. Cells were counted as neurons if they had cell bodies that appeared bright by phase-contrast microscopy, as well as extended cellular processes. Please click here to view a larger version of this figure.
Figure 12. Immunocytochemical identification of cell types in neuronal cultures. The culture condition corresponds to the bottom-left image of Figure 8A. Neurons were obtained from the CA3-CA1 region of the hippocampus of wild-type mice, and plated on a glial feeder layer generated from the CA3-CA1 region of hippocampus of wild-type rats. The cultured cells were analyzed by immunocytochemistry for the neuronal marker MAP2, and the astrocytic glial cell marker GFAP. Differential interference contrast (DIC) microscopy was used to reveal the general morphology of the imaged fields, 14-17 days after the neurons were plated on the glial feeder layer. (A) Three-color staining, including counterstaining with the nuclear marker Hoechst 33342 dye. Two representative image fields are shown. (B) Two-color staining, with different combinations of secondary antibodies conjugated with different fluorophores. In these experiments, the cultured cells were fixed with 4% paraformaldehyde and 4% sucrose in Tyrode's solution (containing, in mM: 125 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 30 D-glucose, 25 HEPES, pH 7.4 adjusted with 5 M NaOH, ~310 mOsm without adjustment) for 30 min at 4 °C. After being washed with Tyrode's solution, they were permeabilized with 0.1% Triton X-100 in Tyrode's solution for 10 min. They were again washed with Tyrode's solution and then blocked with 2% normal goat serum in phosphate-buffered saline (PBS) (blocking solution) for 60 min at RT. Thereafter they were treated with the rabbit polyclonal anti-MAP2 antibody (AB5622; 400x dilution in blocking solution), and mouse monoclonal anti-GFAP cocktail (NE1015; 1,000x dilution) O/N (15-21 hr) at 4 °C. Following washes with PBS, the cells were incubated with goat anti-rabbit and anti-mouse IgG antibody conjugated with Alexa Fluor 405 (500x dilution in blocking solution), Alexa Fluor 488 (1,000x) or Alexa Fluor 568 dye (500x) for 60 min at RT. They were washed with PBS and observed directly in PBS. In some cultures, after the final wash in the above procedures, the cells were treated with Hoechst 33342 at 0.5 µg/ml in PBS for 10 min at RT, and washed with PBS before observation. Please click here to view a larger version of this figure.
Figure 13. Immunocytochemical identification of cell types in glial feeder layer cultures. Sister cultures of the rat glial feeder layers as in Figure 12, but without the plating of mouse neurons. (A) Staining using the immunocytochemical procedures described in Figure 12A. Two representative image fields are shown. (B) Staining of the glial feeder layer using the immunocytochemical procedures described in A, but with the primary antibodies omitted. All MAP2 images in Figures 12A and 13A, B were acquired under the same imaging conditions, and are shown at the same image contrast to allow comparison of intensity. The same procedures were used for GFAP images in Figures 12A and 13A, B. The glial feeder layer culture was imaged on the same day as the cultures in Figure 12. Thus the glial feeder layers in Figures 12 and 13 were cultured in vitro for the same amount of time. Please click here to view a larger version of this figure.
Figure 14. Intensity of immunocytochemical staining. Lines were drawn on the images shown in Figures 12 and 13, and the intensity along these was plotted. Insets show images in the top row of Figure 12A. The three conditions were: 1) plating of mouse cells (containing neurons) on a rat feeder layer and staining with the primary antibodies (Neuron +, glia +, 1° Ab +), 2) glial feeder layer only (no neuronal plating) and staining with the primary antibodies (Neuron −, glia +, 1° Ab +), and 3) glial feeder layer only (no neuronal plating) and staining without primary antibodies (Neuron −, glia +, 1° Ab −). Neuronal staining (MAP2) was present only when mouse cells were plated (conditions 1 vs. 2), and glial staining (GFAP) was present in both sets of cultures (conditions 1 vs. 2). The immunocytochemical staining was specific to both primary antibodies (conditions 1 vs. 3). Please click here to view a larger version of this figure.
SOLUTIONS |
TAE buffer (50x solution) |
Tris base, 486 g |
Glacial acetic acid, 114.2 ml |
0.5 M EDTA, pH 8.0, 200 ml |
Add distilled water to bring total volume to 2 L |
Make up in a chemical fume hood. |
Dilute 50-fold before use. |
Hanks' solution |
Hanks' Balanced Salts, without calcium chloride, magnesium sulfate and sodium bicarbonate (for 1 L) |
NaHCO3, 350 mg (final concentration 4.17 mM) |
HEPES, 2.38 g (final concentration, 10 mM) |
Adjust to pH 7.4 using 5 M NaOH. |
Adjust osmolarity to 310 mOsm using sucrose (Osmolarity tends to ~290 mOsm without any adjustment). |
Add distilled water to bring total volume to 1 L |
Digestion solution (for trypsin treatment of brain tissue) |
NaCl, 800.6 mg (final concentration, 137 mM) |
KCl, 37.3 mg (final concentration, 5 mM) |
Na2HPO4·(7H2O), 187.6 mg (final concentration, 7 mM) |
HEPES, 595.8 mg (final concentration, 25 mM) |
Glucose, 97.3 mg (final concentration, 5.4 mM) |
Adjust to pH 7.2 using 5 M NaOH. |
Osmolarity tends to ~310 mOsm without any adjustment. |
Add distilled water to bring total volume to 100 ml. |
Right before usage, add 20 mg of trypsin (final concentration of 10 mg/ml) and 20 μl of DNase (final concentration of 750 units/ml) to 2 ml of digestion solution. |
Dissociation solution (for mechanical dissociation of brain tissue) |
Hanks' solution |
MgSO4·(7H2O), 295.1 mg (final concentration, 11.97 mM) |
Adjust osmolarity to 310 mOsm using sucrose. |
Total volume is 100 ml. |
Right before usage, add 20 μl of DNase (final concentration of 750 units/ml) to 2 ml of dissociation solution. |
Plating medium-1 (for mouse glial cells) |
Dulbecco's Modified Eagle Medium (DMEM), 449.5 ml |
MITO+ Serum Extender, 0.5 ml |
FBS, 50 ml |
Total volume is 500 ml. |
Plating medium-2 (for mouse neurons) |
Neurobasal-A, 485 ml |
B27, 10 ml |
GlutaMAX-I, 5 ml (final concentration, 2 mM) |
Total volume is 500 ml. |
Plating medium-3 (for rat neurons and glial cells) |
Minimum Essential Media (MEM, without phenol red) |
Glucose, 2.5 g (final concentration, 27.8 mM) |
NaHCO3, 100 mg (final concentration, 2.38 mM) |
Transferrin, 50 mg |
FBS, 50 ml |
GlutaMAX-I, 5 ml (final concentration, 2 mM) |
Insulin, 12.5 mg |
Total volume is 500 ml. |
Growth medium (for rat neurons and glial cells) |
MEM (without phenol red) |
Glucose, 2.5 g (final concentration, 27.8 mM) |
NaHCO3, 100 mg (final concentration, 2.38 mM) |
Transferrin, 50 mg |
FBS, 25 ml |
GlutaMAX-I, 1.25 ml (final concentration, 0.5 mM) |
B27 or NS21 {Chen, 2008 #2399}, 10 ml |
Cytosine β-D-arabinofuranoside (AraC), 0.56 mg (final concentration, 4 µM) |
Total volume is 500 ml. |
General comments |
Our culture media do not contain antibiotics because they could exert cytotoxic effects on cultured glial cells and neurons (reference 70, 71). This makes it especially important to adhere to sterile procedures in culture-related work. |
See the Table of Reagents for the detailed sources of chemicals. |
Table 1. Solutions
The protocol presented here includes procedures for tattooing to label/identify mice, for genotyping mice from tail tips, and for culturing mouse brain neurons at low density. In one round of experiments using 6-8 pups, these procedures typically require ~0.5 hr, ~4 hr and ~2 hr, respectively, at a total of 6-7 hr. This makes it practical for a single experimenter to complete all the procedures necessary from the time of the pups' birth to the plating of neuronal cultures – in less than a single working day (with the exception of prior preparation of glial feeder layers).
Tattooing
Long-term identification of animals is necessary for the purposes of breeding and scientific studies such as analyses of histology, cell function and animal behavior. Tattooing newborn animals is advantageous because it can be carried out rapidly and lasts for much of the animal's life 7,22-25. Tattooing on the paw pads can be better than toe clipping 23 with respect to preserving testable behaviors, for example in suspension or gripping 22,26, although some studies have not noted such deficiencies (e.g., 25). There were no instances of mothers rejecting or cannibalizing pups after tattooing. For other methods of long-term identification of animals, see recent reviews 7,23,24.
Genotyping
A critical feature in our protocol is the use of a fast genotyping method. Although similar genotyping methods were described in previous reports (e.g., 27,28), the system described here has at least two improvements. First, it can tolerate certain variations in the amount of starting tissue, age of animals, and amplicon length. Thus, successful genotyping can be achieved using mice as young as 1 day and as old as 6 months old, and can be carried out using a broad range of primer pairs. Second, this method does not require a stop solution for the DNA extraction step. This eliminates excessive tube handling and pipetting, and allows parallel multi-tube automation of the process with a PCR machine. The number of specimens can be scaled up (e.g., 96 samples) yet processed easily and simultaneously. Also the specimen type is not limited to tail clips; other sample types that could be used include ear punches, toe clippings, whole early embryos, and placenta tissues 2-4,29,30. Different animal species can also be used. Thus the genotyping procedures are reliable (repeatable with low intra-assay variance) and reproducible (low inter-assay variance between runs or between laboratories), and have the advantage of high scalability.
Note that some caution is required for achieving the expected quality of results. First, during DNA extraction, a large variation in the protocol can degrade the results. For example, a significant reduction in the volume of DNA Extraction Solution (e.g., from 200 to 100 µl in step 2.2) still allows genotyping, but it can reduce the reliability and result in variation in PCR results. Under such conditions, it is recommended to reduce the volume of the DNA extract from 4 to 2 µl, and increase the volume of H2O from 2 to 4 µl to compensate for the volume change during PCR reactions (step 2.5). Second, at the end of DNA extraction, the solution can be used for PCR immediately without centrifugation in most cases, although the tail will retain its overall structure and will not be completely decomposed. However, the described inversion of the tubes is essential for the purpose of dissociating genomic DNA from the tissue (step 2.4). It is also important to use the top, clear part of the solution, excluding the debris at the bottom of the tube, because inclusion of the latter will lead to inconclusive PCR results. These steps will be effective with minimal time and effort. Equally good results can be obtained by actively vortexing the specimen (in place of the inversions) followed by centrifugation and usage of the supernatant. Third, after the DNA extraction, the DNA can be stored for long-term (e.g., >two weeks). It is recommended to centrifuge the solution using a microcentrifuge (e.g., 3,000-13,000 g at 4 °C for 2 min), transfer the supernatants to new tubes, and store them at -80 °C. When the stored extract is to be used for genotyping, briefly centrifuge the specimen after thawing, and use the supernatant.
Neuronal cultures
Another critical feature of our protocol is the use of a glial feeder layer to establish neuronal cultures at a low plating density. Typically, in primary cultures of mammalian brain neurons, the cells are plated with a relatively high density, on coated coverslips without a glial feeder layer (e.g., 31-39). When the plating density of neurons is reduced using this method, the initial lack of glial sheet leads to poor neuronal growth and dendritic extension (panels B, C de Figures 8-10), as reported previously 40-42, probably due to poor glial growth 43,44.
Successful, low-density neuronal cultures can be generated by at least three broad types of methods. In one approach, Neurobasal Medium is used as a culture medium. This makes it possible to culture neurons in the absence of a glial feeder layer, and can be used for low-density neuronal cultures 45,46. In a second approach ('Banker-type' and its modification), glial cells are co-cultured with neurons in the same well but are physically separated from them. In this context the glial cells provide 'trophic support' to neurons without contacting them directly 1,41,42,47-49. In the third approach, neurons are plated on a pre-established glial feeder layer that supports neuronal growth (e.g., 50-53) (Figures 8-10). Specifically, mouse brain neurons are plated on a glial feeder layer prepared from mice 8,34,54 or rats 41,55,56.
We prefer the last of the three approaches to low-density culture, because the Neurobasal Medium can affect neuronal survival 57, the astrocytic glial cells will be essential in regulating neuronal functions 8,58, and the physical contact between neurons and glial cells may be important. The procedures for culturing the mouse and rat glial cells are similar 59,60, but we have modified them slightly to accommodate for the more rapid in vitro growth of rat vs. mouse glial cells. The procedures for the rat cell culture were modified from 61-63. The use of a glial feeder layer for low-density neuronal cultures makes it necessary to perform rapid genotyping, in order to match the genotype of the feeder layer to that of the neurons within the period between the pups' births and the neonatal deaths or the end of culture window (1-2 postnatal days).
Low-density culture on a glial feeder layer is also an important method when one tries to culture neurons of small nuclei in the brain (e.g., the norepinephrine-releasing locus coeruleus, and the dopamine-releasing substantia nigra). Those nuclei contain only a small number of neurons and would inevitably yield low-density cultures 64-67.
Primary cultures are routinely prepared in our laboratory from the CA3-CA1 region of hippocampus, the motor region of the cerebral cortex and the striatum of mice. The same procedures can be used to prepare neuronal cultures representing other brain regions, for example the whole hippocampus (including the dentate gyrus) and the whole cerebral cortex. Rat neurons from brain regions such as the hippocampus and the locus coeruleus are cultured in similar fashion, using the rat glial feeder layer. These cultured neurons are usually used at 1-4 weeks in vitro, with the exact age of the culture depending on the purpose of the experiment. It is of note that some neurons cultured on a glial feeder layer can survive for more than 10 weeks 34,68.
One potential problem of low-density neuronal cultures is that the neuronal properties can be different from those in high-density cultures or in neurons in situ. Comparison of these systems provides an interesting opportunity to study how neuronal development and maturation are affected by multiple factors, such as the neuronal density, the soluble factors secreted from neurons, neuron-to-neuron contact, and glial-neuronal interactions.
In summary, the tattooing-based mouse labeling is long lasting, the genotyping method is rapid, and the culture method allows for plating mouse neurons at low density. The protocol described here can be applied in its entirety to other animal species harboring other genetic mutations. Moreover, individual steps of the protocol can be used for other purposes. Thus the protocol can be used in a wide array of applications based on experimenters' needs.
The authors have nothing to disclose.
The authors thank researchers at the University of Iowa, Drs. Luis Tecedor, Ines Martins and Beverly Davidson for instructions and helpful comments regarding striatal cultures, and Drs. Kara Gordon, Nicole Bode and Pedro Gonzalez-Alegre for genotyping assistance and discussions. We also thank Dr. Eric Weyand (Animal Identification and Marking Systems) for helpful comments regarding tattooing, and Dr. Shutaro Katsurabayashi (Fukuoka University) for helpful comments regarding the mouse culture. This work was supported by grants from the American Heart Association, the Department of Defense (Peer Reviewed Medical Research Program award W81XWH-14-1-0301), the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation (N.C.H.).
REAGENTS – tattooing | |||
Machine Cleanser | Animal Identification and Marking Systems, Inc. | NMCR3 | This is used to clean the needles and the holder after tattooing. |
Machine Drying Agent | Animal Identification and Marking Systems, Inc. | NDAR4 | This is used to dry the needles and holder after cleaning. |
Neonate Tattoo Black Pigment | Animal Identification and Marking Systems, Inc. | NBP01 | |
Skin Prep Applicator | Animal Identification and Marking Systems, Inc. | NSPA1 | Q-tip. |
Skin Prep solution | Animal Identification and Marking Systems, Inc. | NSP01 | This reagent delivers a thin layer of oil that enhances the efficiency of tattooing and prevents tattoo fading, by (information from vendor): 1) preventing non-tattooed skin from being stained temporarily, thereby allowing the quality of a paw pad tattoo to be easily evaluated before the pup is returned to its home cage – the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing – softening the skin and lubricating the needle will help the needle penetrate the skin without causing skin damage, and 3) preventing molecular oxygen from entering the skin, thereby reducing inflammatory responses to reactive oxygen species that can be generated. |
REAGENTS – genotyping | |||
EZ Fast Tissue/Tail PCR Genotyping Kit (Strip Tube Format) | EZ BioResearch LLC | G2001-100 | |
2X PCR Ready Mix II | EZ BioResearch LLC | G2001-100 | A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution. |
Tissue Lysis Solution A | EZ BioResearch LLC | G2001-100 | Prepare DNA Extraction Solution by mixing 20 µl of Tissue Lysis Solution A and 180 µl of Tissue Lysis Solution B per specimen. |
Tissue Lysis Solution B | EZ BioResearch LLC | G2001-100 | Prepare DNA Extraction Solution by mixing 20 µl of Tissue Lysis Solution A and 180 µl of Tissue Lysis Solution B per specimen. |
Acetic acid, glacial | VWR | BDH 3092 | |
Agarose optimized grade, molecular biology grade | rpi | A20090-500 | We use 2% agarose gels in TAE buffer containing the SYBR Safe DNA gel stain (diluted 10,000-fold) or ethidium bromide (0.5 µg/ml gel volume). |
Ethidium bromide | Sigma-Aldrich | E7637-1G | |
Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA) | Fisher | BP120-500 | |
Filtered Pipet Tips, Aerosol-Free, 0.1-10 µl | Dot Scientific Inc | UG104-96RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
Filtered Pipet Tips, Premium Fit Filter Tips, 0.5-20 µl | Dot Scientific Inc | UG2020-RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
Filtered Pipet Tips, Premium Fit Filter Tips, 1-200 µl | Dot Scientific Inc | UG2812-RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
Molecular weight marker, EZ DNA Even Ladders 100 bp | EZ BioResearch LLC | L1001 | We use either of these three molecular weight markers. |
Molecular weight marker, EZ DNA Even Ladders 1000 bp | EZ BioResearch LLC | L1010 | |
Molecular weight marker, TrackIt, 100 bp DNA Ladder | GIBCO-Invitrogen | 10488-058 | |
PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml | USA Scientific | 1402-2900 | Use tubes that are sterile and free of DNA, RNase and DNase. An 8-tube strip is easy to handle and to group the specimens than individual tubes. |
PCR tubes, Ultraflux Individual | rpi | 145660 | Use tubes that are sterile and free of DNA, RNase and DNase. |
Seal-Rite 0.5 ml microcentrifuge tube, natural | USA Scientific | 1605-0000 | Use tubes that are sterile and free of DNA, RNase and DNase. |
SYBR Safe DNA gel stain * 10,000x concentration in DMSO | GIBCO-Invitrogen | S33102 | |
Tris base | rpi | T60040-1000 | |
Primers for amplifying Tor1a gene in ΔE-torsinA knock-in mice | 5'-AGT CTG TGG CTG GCT CTC CC-3' (forward) and 5'-CCT CAG GCT GCT CAC AAC CAC-3' (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/µl (~0.16 µM) (reference 2). | ||
Primers for amplifying Tfap2a gene in wild-type mice | 5'-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3' (forward), 5'-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3' (reverse) for the 498-bp amplicon, 5'-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3' (forward), 5'-AGA CAC TCG GGC TTT GGA GAT CAT TC-3' (reverse) for the 983-bp amplicon, and 5'-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3' (forward), 5'-ACA GTG TAG TAA GGC AAA GCA AGG AG-3' (reverse) for the 1990-bp amplicon. These primers are used at 0.5 µM. | ||
REAGENTS – cell culture | |||
5-Fluoro-2′-deoxyuridine | Sigma-Aldrich | F0503-100MG | See comments section of uridine for more information. |
B-27 supplement | GIBCO-Invitrogen | 17504-044 | |
Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treated | BD falcon | 353001 | |
Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm2 Growth Area, 70 ml | BD falcon | 353082 | |
Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm2 Growth Area, 250 ml | BD falcon | 353136 | |
Conical Tube, polypropylene, 15 ml | BD falcon | 352095 | |
Countess (cell number counter) chamber slides | GIBCO-Invitrogen | C10312 | |
Cytosine β-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride) | Sigma-Aldrich | C6645-100mg | |
D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC) | Sigma-Aldrich | G7528-250G | |
Dish, Petri glass 100 x 15 mm | Pyrex | 3160-101 | |
Distilled water | GIBCO-Invitrogen | 15230-147 | |
DNase Type II | Sigma-Aldrich | D4527-200KU | Stock solution is prepared at 1500 units/20 μl = 75000 units/ml in distilled water. |
Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate | GIBCO-Invitrogen | 10569-010, 500 ml | |
Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 µm Pore Size | Nalgene | 568-0020 | |
Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 µm Pore Size | Nalgene | 569-0020 | |
Fetal bovine serum (FBS) | GIBCO-Invitrogen | 26140-079 | |
Glass coverslip, 12 mm Round, thickness 0.09–0.12 mm, No. 0 | Carolina | 633017 | |
GlutaMAX-I | GIBCO-Invitrogen | 35050-061 | |
Hanks' Balanced Salts | Sigma-Aldrich | H2387-10X | |
HEPES, ≥99.5% (titration) | Sigma-Aldrich | H3375-250G | |
Hydrochloric acid, 37%, A.C.S reagent | Sigma-Aldrich | 258148-100 ML | |
Insulin | Sigma-Aldrich | I5500-250 mg | |
Magnesium sulfate heptahydrate, MgSO4•(7H2O), BioUltra, ≥99.5% (Fluka) | Sigma-Aldrich | 63138-250G | |
Matrigel Basement Membrane Matrix solution, Phenol Red-Free | BD Biosciences | 356237 | This is the coating material for coverslips and flasks. 1) To prepare it, thaw the Matrigel Basement Membrane Matrix solution on ice, which usually takes ~1 day. Using a pre-cooled pipette, aliquot the thawed solution into pre-cooled T25 flasks on ice, and store the flasks at -20°C. To prepare the working Matrigel solution, thaw the aliquotted Matrigel in a flask on ice, dilute 50-fold by adding pre-cooled MEM solution and keep the diluted solution at 4°C. It is important to pre-cool all cultureware and media that come into contact with Matrigel, except during and after the coating of coverslips, to prevent it from prematurely forming a gel. 2) To coat the glass coverslips or culture flasks with Matrigel, apply the Matrigel solution to the surface. Before plating cells, it is important to completely dry up the surface. For this purpose, it might be helpful to aspirate Matrigel during the cellular centrifugation immediately before plating the cells and to allow enough time for drying. |
Minimum Essential Medium (MEM) | GIBCO-Invitrogen | 51200-038 | |
MITO+ Serum Extender, 5 ml | BD Biosciences | 355006 | |
Multiwell Plates, Tissue Culture-treated 24-well plate | BD falcon | 353047 | |
Multiwell Plates, Tissue Culture-treated 6-well plate | BD falcon | 353046 | |
Neurobasal-A Medium (1X), liquid | GIBCO-Invitrogen | 10888-022 | |
Nitric Acid | VWR | bdh 3044 | |
NS (Neuronal Supplement) 21 | prepared in the lab | Source: reference 69 | |
Pasteur pipets, 5 ¾” | Fisher | 13-678-6A | Use this cotton-plugged 5 ¾” Pasteur pipette for cellular trituration. Fire-polish the tip beforehand to smooth the cut surface and to reduce the internal diameter to 50-80% of the original. Too small a tip will disrupt the cells and reduce cell viability, but too large a tip will decrease the efficiency of trituration. |
Pasteur pipets, 9” | Fisher | 13-678-6B | |
Potassium chloride (KCl), SigmaUltra, ≥99.0% | Sigma-Aldrich | P9333-500G | |
Serological pipet, 2 ml | BD falcon | 357507 | |
Serological pipet, 5 ml | BD falcon | 357543 | |
Serological pipet, 10 ml | BD falcon | 357551 | |
Serological pipet, 25 ml | BD falcon | 357525 | |
Serological pipet, 50 ml | BD falcon | 357550 | |
Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, ≥99.5% | Sigma-Aldrich | S6297-250G | |
Sodium chloride (NaCl), SigmaUltra, ≥99.5% | Sigma-Aldrich | S7653-250G | |
Sodium hydroxide (NaOH), pellets, 99.998% trace metals basis | Sigma-Aldrich | 480878-250G | |
Sodium phosphate dibasic heptahydrate (Na2HPO4•(7H2O)), ≥99.99%, Aldrich | Sigma-Aldrich | 431478-250G | |
Sucrose, SigmaUltra, ≥99.5% (GC) | Sigma-Aldrich | S7903-250G | |
Syringe filter, sterile, 0.2 µm | Corning | 431219 | |
Syringe, 3 ml | BD falcon | 309585 | |
Transferrin, Holo, bovine plasma | Calbiochem | 616420 | |
Trypan Blue stain, 0.4% | GIBCO-Invitrogen | T10282 | This is used for counting live/dead cells. Renew an old trypan blue solution if it is re-used many times (e.g. several times a week for several weeks), because it will form precipitates and result in erroneous readouts of cellular density. |
Trypsin, type XI | Sigma-Aldrich | T1005-5G | |
Trypsin-EDTA solution, 0.25% | GIBCO-Invitrogen | 25200-056 | |
Uridine | Sigma-Aldrich | U3003-5G | Stock solution is prepared at 50-mg 5-fluoro-2'-deoxyuridine and 125-mg uridine in 25 ml DMEM (8.12 and 20.48 mM, respectively). |
REAGENTS – immunocytochemistry | |||
Antibody, rabbit polyclonal anti-MAP2 | Merck Millipore | AB5622 | |
Antibody, mouse monoclonal anti-GFAP cocktail | Merck Millipore | NE1015 |