This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.
Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.
Human pluripotent stem cell (hiPSC)-derived neurons are increasingly relevant in the areas of basic research, drug development, and regenerative medicine. Workflows and procedures to optimize their culture and maintenance, and improve the efficiency of differentiation into specific neuronal subtypes, are evolving rapidly1,2. To improve the utility and cost-effectiveness of human stem cell-derived neurons as model systems amenable to high-content analyses in drug discovery and target validation, it is useful to decrease the culturing time required to generate mature, functional neurons, while retaining maximum robustness, reproducibility, and phenotype relevance. Although 3-dimensional organoid cultures are driving breakthroughs in neurodevelopment research3, 2-dimensional monolayer cultures are especially compatible with automated imaging-based applications due to their minimal tissue thickness.
However, the adaptation of imaging-based screening methods to models of human neurological and neurodevelopmental disease faces a major challenge. The protracted timeframe over which the human nervous system matures in vivo necessitates extended time in culture to accommodate natural programs of gene expression and achieve neuronal maturation.
One practical consequence of the lengthy neuronal differentiation program is that the maintenance of hiPSC-derived monolayer cultures must be sustained for many weeks to achieve adequate synapse maturity. During this time, neural progenitors that remain undifferentiated continue to divide. These can quickly overgrow the culture and usurp the nutrient content required to maintain viable postmitotic neurons. Vigorously dividing neural progenitor cells (NPCs) can also compete with neurons for the growth substrate. This can render such cultures subject to problems of poor adhesion, a condition unsuitable for imaging-based assays. Moreover, many investigators find that the smaller the culture volume, the greater the difficulty in maintaining healthy populations of differentiated neurons long enough to observe the late stages of neuronal differentiation. In other words, assays of synapse maturation using high content screening (HCS) approaches can be very challenging for human-derived neurons.
To circumvent some of these problems, a procedure of resuspending and replating previously differentiated hiPSC-derived neurons has been used. Firstly, it allows the study of neurite outgrowth (or, more accurately, neurite regeneration) in a population of fully committed neurons. Secondly, the replating of previously differentiated neurons from a large volume format (like 10 cm plates or larger), down to small volume formats (like HCS-compatible 96- or 384-well microtiter plates) enables a significant reduction in total culturing time in the small volume condition. This facilitates the study of synapse assembly and maturation over subsequent weeks in vitro.
However, the replating of mature neurons that have already established long neurites and a complex connectivity network presents several challenges, one of which is the sometimes high and variable rate of cell death. Here, we describe a replating procedure that results in excellent cell survival and reproducibility. Commonly, neurons are exposed to proteolytic enzymes for short incubation periods (typically ~3-10 min) in order to detach cells from the substrate prior to trituration. This brief proteolysis time is customarily used for resuspending and passaging many types of dividing cells, including non-neuronal cells and undifferentiated progenitors4,5,6. However, for differentiated neurons bearing long, interconnected neurites, it is essential not only to detach cells from the substrate but also to disrupt the dendritic and axonal network in order to isolate individual cells while minimizing damage. Indeed, a thick meshwork of neurons usually tends to detach from the substrate as a single sheet, rather than as individual cells. If care is not taken to loosen the thick network of neurites, neurons not only become irreversibly damaged during trituration, but many of them fail to pass through the filter used to remove clumps, resulting in poor cell yield. Below we describe a simple modification to a widely-used protease incubation procedure to counter these difficulties.
In the protocol described below, neurons are incubated for 40-45 min with a mild protease, such as the proteolytic enzyme (e.g., Accutase). During the first 5-10 min after adding the enzyme, the neuronal network lifts off from the substrate as a sheet. Incubation with the protease proceeds for an additional 30-40 min before proceeding with gentle trituration and filtering. This extra incubation time helps ensure that the digestion of the material relaxes the intercellular network, thereby ensuring that subsequent trituration produces a suspension of individual cells. This procedure maximizes the uniformity of cell distribution upon replating while minimizing cell death. We have successfully applied this replating method to hiPSC-derived neuronal cultures generated by various differentiation protocols7,8 and from various lines of hiPSCs. The procedure is nominally suitable for use with most or all lines of stem cell-derived neurons. We have observed that an extended protease incubation time is not absolutely essential for replating cultures from small format plates (e.g., 35 mm diameter); however, as we show here, it provides a significant benefit when replating from large diameter plates (e.g., 10 cm diameter or larger), probably because neurites in such plates can extend very long processes and form a densely interconnected array.
Here we demonstrate this method and briefly illustrate its application in assays for early neuritogenesis and for synapse maturation, which involves clustering of pre- and postsynaptic proteins along the dendrites and axons, followed by their later colocalization at synaptic sites. The examples highlight the advantages this protocol offers in preserving cell viability and reproducibility. First, it permits investigators to study early steps in human neuritogenesis. The experimental setting is similar to the commonly used primary cultures of rodent cortical or hippocampal neurons, where cells are extracted from late fetal or early postnatal brain, dissociated by trituration after gentle protease treatment, and allowed to initiate neurites or to regenerate neurites that were severed in the procedure9,10. Similar to such rodent primary neurons, hiPSC-derived neurons begin to form or regenerate their neurites within hours after replating, allowing imaging of growth cones and neurite morphology in an environment optimal for high spatiotemporal imaging with fewer surrounding undifferentiated cells. We have observed that neurite outgrowth is more synchronized compared to the variable delays and different outgrowth rates seen when neurons first begin to differentiate from a progenitor population. In addition, replating enables assays of neurons expressing neuronal subtype markers that typically appear later in neural development, such as the cortical layer 5/6 transcription factor CTIP2 (Chicken ovalbumin upstream promoter transcription factor-interacting protein 2), or the pan-neuronal marker NeuN11. An especially useful feature of the replating approach is that synaptogenesis proceeds within a time frame compatible with HCS.
1. Differentiation Period Prior to Replating
2. Coating Multiwells
3. Replating Differentiated Neurons
4. Cell Viability Assays Post-replating
5. Immunostaining
6. Calcium imaging
7. Image acquisition and Analysis
NOTE: For details on the acquisition system please refer to Calabrese et al.12.
The replating of hiPSCs-derived neurons that have been differentiated for multiple weeks offers several advantages. However, detaching and replating differentiated neurons that have long, interconnected dendrites and axons (Figure 1A) can result in a high fraction of irreversibly damaged neurons.
As described in the protocol section, we used incubation with a proteolytic enzyme to detach the neurons from the substrate. Typically, due to their thick meshwork of neurites (Figure 1A), the cells tend to detach all at once as a single syncytial-like layer, which often begins to float in the well (Figure 1C). This happens fairly quickly, and is perhaps why most laboratories tend to collect the cells after only 5 min of incubation with their proteolytic enzyme of choice, and to immediately break apart the sheet of cells by trituration (pipetting it up and down). However, this mechanical manipulation appears to result in high stress for neurons. We believe the degree of stress may be proportional to the area covered by the neuronal network, because we find that the apparent damage from inadequate protease incubation is greater for 10 cm or larger plates than for 35 mm or smaller plates. Thus, counterintuitively, we found that prolonging the enzymatic incubation to 40-45 min reduces cell death at the time of cell dissociation (Figure 1C) and permits more efficient recovery of live, healthy cells that emit processes over hours to days post-replating (Figure 1B). Presumably, the extended incubation time with a mild proteolytic enzyme allows the partial digestion of proteins that strongly adhere cells and their processes to one another and to the extracellular matrix. This allows the resuspension process to work efficiently to separate individual cells without the need for overly vigorous trituration.
To quantify the effectiveness of the extended enzyme incubation procedure, we evaluated viability of suspended cells immediately after trituration using trypan blue (Figure 1C), and later at 1, 3 and 7 days post-replating using an early cell death marker (Figure 2). Immediately after trituration, trypan blue staining indicated that there was substantially greater cell death after a 5 min incubation compared to a 45 min incubation with the proteolytic enzyme (Figure 1C). The two iPSC lines we used to illustrate this observation were differentiated into neurons from the neural progenitor stage using different protocols, and showed broadly different sensitivity to the replating procedure. Cultures from the WT126 line differentiated into NPCs using the "rosette selection method"7 were more sensitive to damage than cultures differentiated from the CVB WT24 line using a rapid differentiation method8. Nevertheless, for both lines the degree of immediate cell death was approximately halved by using the extended enzyme incubation procedure.
After replating, cultures exhibited an approximate doubling of cell viability over subsequent days using the extended enzyme incubation procedure, as determined by the density of DAPI-positive cells (Figure 2B, graph on the left). In addition, the extended enzyme incubation resulted in a lower density of dead or dying cells detected using a dye-exclusion viability kit (Figure 2B, graph on the right). The fraction of dead cells detected using the viability assay after 24 h post-replating was higher than that seen using trypan blue immediately following trituration. This probably reflects the accumulation of dead and dying cells over these first 24 h, although it is also possible that the viability assay more sensitively detects cell death than the trypan blue assay. Dead cells continued to be detected over 1-7 days post-replating (Figure 2B). The initial plating density for both experimental groups (5 min and 45 min) was identical and based on hemocytometer live cell counts of the post-trituration cell suspension. Importantly, at all time points post-replating, the number of live, healthy cells was approximately doubled following the 45 min enzyme incubation compared to the 5 min incubation. These observations were confirmed qualitatively using phase contrast microscopy to monitor cell morphology at every step: before replating, during replating, and after replating (Figure 1 and Figure 2).
One of the advantages of replating hiPSC-derived neurons after they have been differentiating for several weeks is that most of the cells will have exited the progenitor stage and become committed to a neuronal phenotype at the time of replating. The transition phase of neuronal differentiation from neural progenitors takes place over several days, with greater numbers of cells gradually expressing early-stage neuronal markers, such as beta-III tubulin (detected by antibody TuJ1) or MAP2. Gene expression evolves over several weeks, eventually resulting in expression of late-stage pan-neuronal markers, such as NeuN, or cell-type specific markers for cortical neurons such as CTIP2. Therefore, the replating of previously differentiated hiPSC-derived neurons allows one to study early and transient neuronal events, such as neurite initiation, in identified subtypes of neurons. Figure 3 illustrates the immediate presence of early neuronal markers in cultures that were replated after 4 weeks of pre-differentiation in a larger culture dish. Note that NeuN- and CTIP2-expressing neurons are readily identified within a few days post-replating (Figure 3). Characteristics and timing of neuronal differentiation can vary among hiPSC lines. For the WT 126 line and the CVB WT24 lines we describe here, 85 ± 12% (n = 2) and 52 ± 11% (n = 5) of the cells, respectively, expressed immunoreactivity for the βIII-tubulin marker TuJ1 at 4 days after replating using the extended protease incubation procedure. For both lines, 30-40% of the cells, and 80-90% of the neurons also express the layer 5/6 neocortical marker CTIP2. In addition to improved viability, the extended protease protocol also moderately enhanced neurite outgrowth (average neurite length per neuron), as shown in Figure 4. Both total neurite length (quantified using antibody Tuj1, which stains both axons and dendrites; Figure 4B, left graph) and dendrite growth (quantified using MAP2, which stains only dendrites; Figure 4B, center graph) were favored when using the extended protease incubation procedure. Note that the graph for DAPI-positive (DAPI (+)) cell counts in Figure 2 are based on the same image samples as the cell count graph in Figure 4, but in Figure 2 they were quantified using a non-automated method assisted by Fiji software, while in Figure 4 they were quantified using the automated image analysis software platform CellProfiler. These two image analysis approaches yielded similar results.
Replating is useful not only to study neurite outgrowth, but also to study synapses or other later-appearing biological features of neurons. Indeed, after just one week post-replating we observe markers for many presynaptic and postsynaptic proteins decorating the neuronal dendrites in a punctate pattern (Figure 5A). After 4 weeks we also begin to detect their colocalization, which is an indicator of the formation of functional synapses (Figure 5A). Moreover, electrical activity from spontaneous depolarization and synaptically driven currents is detectable using calcium imaging (Figure 5B) or multielectrode arrays (MEAs).
Figure 1: Superior preservation of neuronal viability after longer incubation with the proteolytic enzyme for cell dissociation. (A) Phase contrast image of NPC-derived neurons differentiated for 3 weeks. The boxed region in the left image is enlarged at right. At this stage neurons have long processes, which form a thick meshwork. Scale bars = 80 µm (left); 35 µm (right). (B) Selected images of hiPSC-derived neurons at 1 and 5 days post-replating. Scale bar = 100 µm. (C) Left: cells detach as a single sheet within a few minutes after initiating protease treatment. Scale bar = 200 µm. Right: after trituration cells are counted on the hemocytometer before replating. Graphs show quantification of non-viable, trypan-blue positive cells after 5 min or 45 min incubation with the proteolytic enzyme for two different lines CVB WT24 (*** p < 0.0003, unpaired t-test) and WT 126 (*** p < 0.0001, unpaired t-test). Values represent the mean ± S.E.M of 4 individual replicates. Please click here to view a larger version of this figure.
Figure 2: Neuronal viability after several days post-replating. (A) Images of WT126 NPC-derived neurons at 1 and 3 days post-replating using 5 min and 45 min protease incubation. Dying cells are labeled with the sensitive early cell death reporter. Corresponding brightfield images are shown on the left. Some cellular debris (blue arrow) is typically detected after replating, especially in the 5 min group. Scale bars = 150 µm. (B) Images of CVB WT24 NPC-derived cultures at 1, 3 and 7 days post-replating using 5 min and 45 min protease incubation. Dying cells are labeled with the sensitive early cell death reporter (red). All nuclei of living and dying cells are labeled with DAPI (cyan). The merged white signal represents DAPI and VivaFix-positive (+) cells. A few cells display VivaFix staining but lack DAPI staining (red cells in the combined image on the right); these are likely cells that were dead for many hours. Scale bars: 25 µm. Right graph shows quantification of the fraction of dead cells (VivaFix/DAPI) after different incubation time with the proteolytic enzyme (5 min vs 45 min: * p < 0.05 1 d and *** p < 0.001, 3 d; two-way ANOVA, followed by multicomparison Bonferroni post hoc test). Left graph shows changes in overall cell density (# DAPI(+) cells for 5 min vs 45 min: *** p < 0.0001 for all time points; two-way ANOVA, followed by multicomparison Bonferroni post hoc test). All values are shown as the mean ± S.E.M of 3 replicates, with a minimum of 1,500 cells scored per condition. Please click here to view a larger version of this figure.
Figure 3: Detectable expression of late-stage neuronal markers immediately after replating. Cultures of CVB WT24 hiPSC-derived cells imaged 4 days after replating, using 45 min protease incubation, from a 10 cm dish that had been differentiated from the NPC stage for 4 weeks. Examples shown were stained for the pan-neuronal markers TuJ1, MAP2, or NeuN; or the deep-layer cortical pyramidal neuron marker CTIP2. Note the presence of extensive neurites, and the robust expression of TuJ1 and MAP2. Note especially that a substantial fraction of the neurons also expresses late-stage markers NeuN and CTIP2. Scale bar = 16 µm. Please click here to view a larger version of this figure.
Figure 4: Neurite and dendrite growth over time following shorter and longer enzyme incubation during replating. (A) Replated CVB WT24 hiPSC-derived neurons quickly extend neurites, as detected using antibody Tuj1. Note that the extended protease incubation time promotes neuritogenesis. Scale bar = 25 µm. (B) Quantification of neurite and dendrite length, over 1, 3 and 7 days post-replating using either 5 min or 45 min protease. Total neurite length was quantified from TuJ1 (βIII-tubulin) staining; dendrite-specific staining was quantified from MAP2 staining snf corrected for the overall cell density (right graph). All values are shown as the mean ± S.E.M of 3 replicates. Neurite length 5 min vs 45 min: * p < 0.05 at 1 day and 7 days, ** p < 0.01 at 3 day; dendrite length 5 min vs 45 min: ** p < 0.01 at 1 day and 3 days, not significant (ns) at 7 day; # DAPI(+) 5 min vs 45 min: *** p < 0.0001 for all time points; two-way ANOVA, followed by multicomparison Bonferroni post hoc test. Please click here to view a larger version of this figure.
Figure 5: Synaptogenesis and spontaneous calcium transients in differentiated hiPSC-derived neurons after replating. (A) Left: at 4 weeks post-replating using the extended protease incubation protocol, the presynaptic marker synapsin 1 is detectable in punctate clusters along the MAP2 positive dendrites. Scale bars = 5 µm. Right: selected dendritic region showing colocalization between presynaptic and postsynaptic clusters (yellow arrow), an indication of potentially active synapses. Scale bars = 1.5 µm. (B) Left: hiPSC-derived neurons infected with AAV8-syn-jGCaMP7f-WPRE were used to monitor spontaneous calcium transients driven by network activity. The brightfield image is shown adjacent to the pseudocolor rendering of GCaMP7 fluorescence at a single time point in the time-lapse series. Scale bar = 25 µm. Colored numbers indicate the 4 selected cells in which GCaMP7 fluorescence was measured over time, as shown in the traces on the right. Each colored trace corresponds to one cell. The asterisk points to the time during the live recording to which the image on the left corresponds. Please click here to view a larger version of this figure.
Figure 6: Workflow of replating procedures to culture hiPSCs for high content screening and/or detailed studies of differentiation and neurite outgrowth or MEA recordings. Starting from a differentiated culture of neurons grown for 4 or more weeks in a larger format (e.g., a 10 cm culture dish), neurons are incubated with a protease for 40-45 min, triturated gently to dissociate and resuspend. Cells are then distributed into multi-wells compatible with HCS, replated onto substrates compatible with imaging growth cones (yellow arrow) or other structures, or onto multi-wells suitable for multielectrode array recordings. Scale bars = 27 µm, 5 µm, 2.5 µm, 130 µm (from left to right). Please click here to view a larger version of this figure.
We have demonstrated a straight-forward procedure for the resuspension and replating of human neuronal cultures that optimizes viability, differentiation success, and subcellular imaging in a manner that is compatible with high content screening platforms, and other assays relevant to drug discovery. Figure 6 illustrates the overall workflow and examples of such applications.
Although here we focused on hiPSC-derived neurons that are directed toward a cortical neuron fate, we expect that this method should be equally applicable to human embryonic stem cell-derived neurons, and to stem-cell derived neurons directed toward other neuronal phenotypes14,15,16,17,18. Moreover, we postulate that this extended protease procedure might be beneficial to other situations that require resuspension of cells having an established neurite meshwork, for example for FACS sorting or single-cell analyses of primary neurons19,20.
Replating hiPSC-derived neurons provides a viable model system in which to investigate cellular and molecular mechanisms of many key biological events. For example, this procedure can facilitate detailed studies of human neurite outgrowth and growth cone characteristics, and comparison of findings to the extensive knowledge base built from decades of studying other species, both vertebrate and invertebrate. We find that the replating procedure has made it is easier to optimize conditions to generate larger growth cones that are well-suited for optical evaluation of subcellular structure and function (Figure 6). By comparison, the growth cones more typically observed during the initial differentiation of neurons from hiPSCs tend to be small and compact, and the dense array of non-neuronal cells in such cultures makes it difficult both to adjust substrate conditions to promote growth cone spreading and to image individual growth cones within the complex tissue environment. Thus, replating neurons onto a fresh dish provides a "cleaner slate" on which to view growth cones under good imaging conditions.
Replating is particularly advantageous when using cell culture platforms suitable for HCS because it greatly reduces the total time in which cultures are grown in small-volumes. The small working volumes of the individual wells of 96, 384 or 1536-well multi-wells (approximately 100, 50 and 5 microliters working volume, respectively) usually requires frequent (i.e., daily) media changes to counteract evaporation and nutrient depletion. Frequent media changes are costly, not only in terms of reagents and labor, but they can compromise the viability of cultures by diluting conditioned media and by increasing the chances that cells detach from the substrate due to mechanical turbulence. Replating of hiPSCs can also facilitate the recording of physiological activity using multielectrode arrays21,22, or live imaging of cultures in assays of dynamic neuronal phenotypes23.
The authors have nothing to disclose.
This work is a component of the National Cooperative Reprogrammed Cell Research Groups (NCRCRG) to study mental illness and was supported by NIH grant U19MH107367. Initial work was also supported by NIH grant NS070297. We thank Drs. Carol Marchetto and Fred Gage, The Salk Institute, for providing the WT 126 line of neural progenitor cells, and Drs. Eugene Yeo and Lawrence Goldstein, UC San Diego for providing the CVB WT24 line of neural progenitor cells. We thank Deborah Pre in the laboratory of Dr. Anne Bang, Sanford Burnham Prebys Medical Discovery Institute, for useful discussions.
Post Replating Media | |||
L-Ascorbic Acid | Sigma | A4403 | Add 1ml of 200mM stock to 1L of N2B27 media |
dibutyryl-cAMP | Sigma | D0627 | Add 1 µM |
Human BDNF | Peprotech | 450-02 | 10 ng/ml final concentration |
B27 (50X) | Thermofisher Scientific | 17504044 | Add 20 ml to 1L N2B27 media |
DMEM/F12 with Glutamax | Thermofisher Scientific | 31331093 | Add N2 and distribute in 50 mL conicals; parafilm wrap lids |
Human GDNF | Peprotech | 450-10 | 10 ng/ml final concentration |
Glutamax | Thermofisher Scientific | 35050038 | Add 10 ml to 1L N2B27 media; glutamine supplement |
Mouse Laminin | Sigma | P3655-10mg | Add 100 µl to 50 mL N2B27 |
MEM Nonessential Amino Acids | Thermofisher Scientific | 11140035 | Add 5ml to 1L N2B27 media |
N2 (100X) Supplement | Life Technologies | 17502048 | Add 5ml to 500mL media |
Neurobasal A Media | Thermofisher Scientific | 10888022 | Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media |
Neurobasal Media | Thermofisher Scientific | 21103049 | for WT126 cells; neural basal media |
SM1 Supplement | StemCell Technologies | 5711 | Add 1:50 to media |
sodium bicarbonate | Thermofisher Scientific | 25080-094 | Add 10ml to 1L N2B27 media |
Plate Preparation | |||
10cm Tissue Culture Dishes | Fisher Scientific | 08772-E | Plastic TC-treated dishes |
6-well Tissue Culture Dishes | Thomas Scientific | 1194Y80 | NEST plates |
Mouse Laminin | Life Technologies | 23017-015 | Add 1:400 on plastic |
Poly-Ornithine | Sigma | P3655-10mg | Add 1:1000 on plastic |
UltraPure Distilled Water | Life Technologies | 10977-015 | To dilute Poly-L-Ornithine |
Replating Reagents | |||
100mM Cell Strainer | Corning | 431752 | Sterile, individually wrapped |
384-well plate, uncoated | PerkinElmer | 6007550 | Coat with PLO and Laminin |
DPBS | Life Technologies | 14190144 | Dulbecco's phosphate-buffered saline |
Poly-D-Lysine-Precoated 384-well Plates | PerkinElmer | 6057500 | Rinse before coating with laminin |
StemPro Accutase | Life Technologies | A1110501 | Apply 1mL/10cm plate for 30-45 minutes; proteolytic enzyme |
Fixation Materials | |||
37% Formaldehyde | Fisher Scientific | F79-1 | Dissolved in PBS |
Sucrose | Fisher Scientific | S5-12 | 0.8 g per 10 ml of fixative |
Immunostaining Materials | |||
Alexa Fluor 488 Goat anti-mouse | Invitrogen | A-11001 | secondary antibody |
Alexa Fluor 568 Goat anti-chicken | Invitrogen | A-11041 | secondary antibody |
Alexa Fluor 647 Goat anti-chicken | Invitrogen | A-21449 | secondary antibody |
Alexa Fluor 561 Goat anti-rat | Invitrogen | A-11077 | secondary antibody |
DAPI | Biotium | 40043 | visualizes DNA |
mouse antibody against b3-tubulin (TuJ-1) | Neuromics | MO15013 | early stage neuronal marker |
rat antibody against CTIP2 | Abcam | ab18465 | layer 5/6 cortical neurons |
chicken antibody against MAP2 | LifeSpan Biosciences | LS-B290 | early stage neuronal marker |
chicken antibody against NeuN | Millipore | ABN91 | late stage neuronal marker |
rabbit antibody against MAP2 | Shelley Halpain | N/A | early stage neuronal marker |
mouse antibody against PSD-95 | Sigma | P-246 | post-synaptic marker |
rabbit antibody against Synapsin 1 | Millipore | AB1543 | pre-synaptic marker |
Bovine serum albumin (BSA) | GE Healthcare Life Sciences | SH30574.02 | 10% in PBS for blocking |
Titon X-100 | Sigma | 9002931 | Dilute to 0.2% on PBS for permeabilization |
Viability Markers | |||
Vivafix 649/660 | Biorad | 135-1118 | cell death marker |
Calcium Imaging | |||
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
AAV8-syn-jGCAMP7f-WPRE | THE SALK INSTITUTE, GT3 Core Facility | N/A | calcium reporter in a viral delivery system |
hiPSC-derived NPCs | |||
WT 126 (Y2610) | Gage lab | N/A | Marchetto et al., 2010 |
CVB WT24 | Yeo and Goldstein labs | N/A | unpublished |