NeuroCult™ SM1 Neuronal Supplement

Serum-free supplement for the culture and differentiation of neural cells, optimized from B27 formulation

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NeuroCult™ SM1 Neuronal Supplement

Serum-free supplement for the culture and differentiation of neural cells, optimized from B27 formulation

10 mL
Catalog #05711
93 USD

Required Products

Overview

NeuroCult™ SM1 Neuronal Supplement is a standardized serum-free supplement for the culture of mouse and rat primary neurons and the differentiation and maturation of human embryonic stem cell/induced pluripotent stem cell-derived neurons. NeuroCult™ SM1 can also be used as a versatile serum-replacement supplement for various applications. The formulation of NeuroCult™ SM1 Neuronal Supplement is based on the published B27 formulation (Brewer et al.), but has been optimized to more reproducibly support increased survival and maturation of functional neurons in both short- and long-term culture. This supplement may be used in combination with BrainPhys™ Neuronal Medium or NeuroCult™ Neuronal Basal Medium.

Also available: NeuroCult™ SM1 Without Vitamin A.
Advantages:
• Formulated to support improved cell survival in long-term primary neuronal culture
• Cultures feature increased neurite outgrowth and branching in short- and long-term cultures
• Product undergoes rigorous performance testing
Contains:
• Antioxidants
• Vitamin A
• Insulin
• Other ingredients
Subtype:
Supplements
Cell Type:
Neurons; Pluripotent Stem Cells; Neural Cells, PSC-Derived
Species:
Human; Mouse; Rat
Application:
Cell Culture; Maintenance; Differentiation
Brand:
NeuroCult
Area of Interest:
Neuroscience; Stem Cell Biology
Formulation:
Serum-Free; Defined

Scientific Resources

Educational Materials

(13)
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Data and Publications

Data

Morphology of Neurons in Representative NeuroCult™ SM1 Cultures at 7 and 21 Days in Vitro

Figure 1. Morphology of Neurons in Representative NeuroCult™ SM1 Cultures at 7 and 21 Days in Vitro

Primary rat E18 cortical neurons were cultured for 7 (A) and 21 (B) DIV in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium. (A) Phase contrast imaging at 7 DIV shows large numbers of viable neurons, with minimal cell clumping and extensive neurite outgrowth and branching. (B) After 21 DIV, large numbers of viable neurons with developed dendritic arbors remain in culture. Magnification 20x.

Number of Neurons in NeuroCult™ SM1 and TSFM Cultures After 7 and 21 Days in Vitro

Figure 2. Number of Neurons in NeuroCult™ SM1 and TSFM Cultures After 7 and 21 Days in Vitro

(A) Comparable numbers of neurons are obtained when cells are cultured for 7 days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 25; mean ± 95% CI; p > 0.05). (B) Significantly higher numbers of neurons are obtained when cells are cultured for 21 days in a NeuroCult™ SM1-supplemented medium compared to TSFM (n = 25; mean ± 95% CI; ***p < 0.001).

Neurite Outgrowth of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Figure 3. Neurite Outgrowth of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Significantly longer neurite outgrowth was observed for cells cultured for 7 (A) and 21 (B) days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 240 independent measures, mean ± 95% CI, ***p < 0.001).

Neurite Branching of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Figure 4. Neurite Branching of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Significantly more neurite branch points were observed for cells cultured for 7 (A) and 21 (B) days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 240 independent measures, mean ± 95% CI, ***p < 0.001).

Expression of Pre- and Post-Synaptic Markers in Neurons Cultured for 21 Days in NeuroCult™ SM1

Figure 5. Expression of Pre- and Post-Synaptic Markers in Neurons Cultured for 21 Days in NeuroCult™ SM1

Neurons cultured in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium for 21 days are phenotypically mature as indicated by the presence of an extensive dendritic arbor and appropriate expression and localization of pre- (Synapsin) and post-synaptic (PSD-95) markers. (A-C) Synapsin (green) staining is concentrated in discrete puncta distributed along the somata and dendritic processes, as defined by MAP2 (red) staining. (D-F) Dendritic staining observed for MAP2 and punctate staining for the postsynaptic marker PSD-95. Nuclei were counter-stained with DAPI. Scale bar 10 µm.

Publications

(9)
PloS one 2017 JAN

Development of a DIPG Orthotopic Model in Mice Using an Implantable Guide-Screw System.

Marigil M et al.

Abstract

OBJECTIVE In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system. METHODS It consisted of a guide-screw also called bolt, a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy, antibodies or gene/viral therapies. RESULTS The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model. CONCLUSION Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons.
Nature Communications 2016 MAR

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

Chamma I et al.

Abstract

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.
Nature communications 2015 SEP

Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5.

Brigidi GS et al.

Abstract

Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled, in part, through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase, DHHC5, to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions, DHHC5 is bound to PSD-95 and Fyn kinase, and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast, DHHC5's substrate, δ-catenin, is highly localized to dendritic shafts, resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes, enhancing DHHC5 endocytosis, its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin, DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane.
Neurobiology of Aging 2015 FEB

Aβ and NMDAR activation cause mitochondrial dysfunction involving ER calcium release

Ferreira IL et al.

Abstract

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aβ) peptide. Aβ interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aβ, we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aβ + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization, compared with Aβ or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aβ or NMDA, suggesting that mitochondria control Ca(2+) entry through NMDARs. However, incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aβ. Aβ-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil, an antagonist of GluN2B-containing NMDARs. Exposure to Aβ + NMDA further evoked a higher mitCa(2+) retention, which was ameliorated in GluN2B(-/-) cortical neurons, largely implicating the involvement of this NMDAR subunit. Moreover, pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aβ + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether, data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aβ-mediated GluN2B-composed NMDARs activation.
Cell Death and Disease 2015 FEB

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons

Curcio M et al.

Abstract

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.
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