NeuroXVivo Rat Cortical Neuron Culture Kit CDK011: R&D Systems
Kit Summary

Reagents for the maintenance and maturation of rat cortical neurons in culture.

Key Benefits

  • Features an optimized media for rat cortical neuron cultures
  • Includes R&D Systems® Recombinant Human BDNF and IGF-I
  • Designed for culturing of either embryonic or postnatal cortical neurons
  • Simple and reliable procedures
  • Ideal for both non-experienced and experienced neural cell culture researchers.
 

 

Why use the NeuroXVivo Rat Cortical Neuron Culture Kit to mature rat cortical neurons in vitro?

Cortical neuron culture is an important model system for studies of neuronal development and function, neurotoxicity screening, drug discovery, and mechanisms of neurological diseases. Proper development and survival of neurons, in vivo as well as in vitro, requires specific growth factors, signaling molecules, peptides, and vitamins. The NeuroXVivo Rat Cortical Neuron Culture Kit provides optimized reagents and growth factors, as well as a validated protocol, to successfully culture rat cortical neurons in a serum-free environment. The kit contains a Neuronal Base Media, a neuronal media supplement, and growth factors to support the robust growth and maintenance of both short- and long-term neuronal cultures. This kit is designed to work with primary rat cortical neurons isolated during either embryonic or postnatal stages. The NeuroXVivo Rat Cortical Neuron Culture Kit is a simple and reliable tool for culturing primary rat cortical neurons for both experienced and non-experienced users.

NeuroXVivo Rat Cortical Neuron Culture Kit:

  • Features an optimized media for rat cortical neuron cultures
  • Includes R&D Systems® Recombinant Human BDNF and IGF-I
  • Designed for culturing of either embryonic or postnatal cortical neurons
  • Simple and reliable procedures
  • Ideal for both non-experienced and experienced neural cell culture researchers.
 

 

Kit Contents

This kit contains the following reagents to maintain mouse pluripotent stem cells in culture:

  • Neuronal Base Media (500 mL)
  • N21-MAX Media Supplement (50X)
  • Recombinant Human BDNF
  • Recombinant Human IGF-I
  • Reconstitution Buffer 1

Stability and Storage

Store unopened kit at < -20 °C in a manual defrost freezer. Do not use past the expiration date.

Limitations

  • FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
  • The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
  • This reagent should not be used beyond the expiration date indicated on the label.
  • Results may vary due to variations among cells derived from different donors.
 

 

Data Examples
Enhanced Neurite Development and Branching using the NeuroXVivo Rat Cortical Neuron Culture Kit Compared to Competitor.
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Enhanced Neurite Development and Branching using the NeuroXVivo Rat Cortical Neuron Culture Kit Compared to Competitor. Primary cortical neurons from either E18 or P1 rat pups were plated at a medium density and cultured using (A, C) the NeuroXVivo Rat Cortical Neuron Culture Kit (R&D Systems, Catalog # CDK011); or (B, D) neural media and supplements from the most widely used competitor. After 14 days in vitro, neurons were stained with Neuron-specific-beta-III Tubulin Northern Lights (NL) 493-conjugated Antibody (R&D Systems, Catalog # NL1195G; green) and counterstained with DAPI (Tocris, Catalog # 5748; blue). E18 rat cortical neurons cultured with the NeuroXVivo Rat Cortical Neuron Kit (A) show more extensive neurite outgrowth compared to E18 cortical neurons cultured with competitor supplements (B). P1 rat cortical neurons cultured with the NeuroXVivo Rat Cortical Neuron Kit (C) also show more extensive neurite outgrowth compared to E18 cortical neurons cultured with competitor supplements (D).

More Robust Synaptic Puncta Development for Primary Rat Cortical Neurons Cultured using NeuroXVivo Rat Cortical Neuron Culture Kit.
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More Robust Synaptic Puncta Development for Primary Rat Cortical Neurons Cultured using NeuroXVivo Rat Cortical Neuron Culture Kit. E18 primary rat cortical neurons were plated at a medium density and cultured using (A) the NeuroXVivo Rat Cortical Neuron Culture Kit (R&D Systems, Catalog # CDK011), or (B) neural media and supplements from the most widely used competitor. After 14 days in vitro, synaptic puncta were visualized using a Rat Synaptotagmin-1 Monoclonal Antibody (R&D Systems, Catalog # MAB4364), followed by the NorthernLights (NL)557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (R&D Systems, Catalog # NL007; red). Cells were counterstained with DAPI (Tocris, Catalog # 5748; blue). Cortical neurons cultured with the NeuroXVivo Cortical Neuron Culture Kit show more robust synaptic puncta development and dendritic arborization, compared to the competitor.

Depolarization Mediated Intracellular Calcium Response in Cultured Rat Cortical Neurons.
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Depolarization Mediated Intracellular Calcium Response in Cultured Rat Cortical Neurons. E18 rat cortical neurons cultured in using the NeuroXVivo® Rat Cortical Neuron Culture Kit for 14 days in vitro were depolarized with either of (A) 3 µM glutamate, (B) 50 mM potassium chloride, or (C) HBSS control. Depolarization was monitored by tracking changes in intracellular Ca2+ levels (F340/F380) using Fura-2. Representative histograms are shown for each depolarization condition.

Preparation and Storage
  • Stability & Storage
    Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, primary rat cortical neurons are maintained and matured ex vivo with the NeuroXVivo Rat Cortical Neuron Culture Kit using the following procedure:

  • Dissect prenatal or neonatal rat cortical tissue
  • Dissociate rat cortical tissue
  • Suspend single cells in Complete Cortical Neuron Culture Media
  • Plate cells onto coated plates
  • Exchange media every 3-4 days
 

 

Reagents Provided

Reagents provided in the NeuroXVivo Rat Cortical Neuron Culture Kit (Catalog # CDK011)

  • Neuronal Base Media (500 mL)
  • N21-MAX Media Supplement (50X)
  • Recombinant Human BDNF
  • Recombinant Human IGF-I
  • Reconstitution Buffer 1

 

Other Supplies Required

Reagents

  • E17–E18 Timed Pregnant Rat or P1-P2 Rat Pups
  • Poly-D-Lysine (R&D Systems®, Catalog # 3439-100-01)
  • Poly-L-Lysine –coated µ-slides
  • Mouse Laminin-I (R&D Systems®, Catalog # 3400-010-01)
  • L-Glutamine
  • Antibiotic-Antimycotic (100X)
  • Phosphate Buffered Saline (PBS)
  • Sterile deionized or distilled water (dH2O)
  • Papain
  • DNAse-I
  • Ovomucoid Protease Inhibitor
  • Earle’s Balanced Salt Solution

Materials

  • Parafilm
  • Fire-polished glass pasteur pipette
  • Tissue culture plates
  • Conical tubes
  • Pipettes and pipette tips

Equipment

  • 37 °C, 5% CO2 humidified incubator
  • Laminar flow cell culture hood
  • Centrifuge
  • Hemocytometer
  • 37 °C water bath
  • Microscope

 

Reagent Preparation

Neuronal Base Media - Thaw at 37 °C.

 

N21-MAX Supplement (50X) - Thaw at 2-8 °C.

 

Recombinant Human BDNF (1000X) - Add 560 µL of Reconstitution Buffer 1 to the vial of Recombinant Human BDNF to produce Recombinant Human BDNF (1000X).

 

Recombinant Human IGF-I (1000X) - Add 600 µL of Reconstitution Buffer 1 to the vial of Recombinant Human IGF-I to produce Recombinant Human IGF-I (1000X).

 

Complete Cortical Neuron Culture Media - Add N21-MAX Supplement (50X) at a final concentration of 1X to the desired amount of Neuronal Base Media (e.g., for every 100 mL of base media, add 2 mL of N21-MAX Supplement (50X)). Add Recombinant Human BDNF (1000X) and Recombinant Human IGF-I (1000X) at a final concentration of 1X to the desired amount of Neuronal Base Media (e.g., for every 100 mL of base media, add 100 µL each of Recombinant Human BDNF (1000X) and Recombinant Human IGF-I (1000X)). Supplement media with 0.5 mM L-Glutamine and Antibiotic-Antimycotic (1X).

 

Note: Complete Cortical Neuron Culture Media is stable for up to 1 month at 2-8 °C after adding all the growth supplements.

 

 

Procedure Overview

Please refer to the product datasheet for complete protocol details.

Note: Optimal culture conditions for each stem cell line must be determined by the investigator.

Day 1 Prepare tissue culture plates by coating with Poly-D-Lysine and Laminin-I.

Day 1 Prepare tissue culture plates by coating with Poly-D-Lysine and Laminin-I.

Day 2 Isolate cortical tissue from E16-18 rat embryos or P1-2 pups following the dissection protocol outline.

Day 2 Isolate cortical tissue from E16-18 rat embryos or P1-2 pups following the dissection protocol outline.

Digest cortical tissue with Papain and DNase-1 for 20-30 minutes.*
*Only if using brains from postnatal rats.

Digest cortical tissue with Papain and DNase-1 for 20-30 minutes.

Collect tissues in a conical tube containing Neuronal Base Media and dissociate into a single-cell suspension using a fire-polished pasteur pipette.

Collect tissues in a conical tube containing Neuronal Base Media and dissociate into a single-cell suspension using a fire-polished pasteur pipette.

Pellet the cells by centrifugation at 200 x g. Suspend cells in Neuronal Base Media and pellet cells. Repeat a total of two times.

Pellet the cells by centrifugation at 200 x g. Suspend cells in Neuronal Base Media and pellet cells.

Suspend cell pellet in Complete Cortical Neuron Culture Media. Perform a cell count.

Suspend cell pellet in Complete Cortical Neuron Culture Media.

Verify desired amount of cells per milliliter in Complete Cortical Neuron Culture Media.

 

Seed cells onto Poly-D-Lysine/ Laminin-I coated tissue culture plates or µ-slides.

Verify desired amount of cells per milliliter in Complete Cortical Neuron Culture Media.

Culture cortical neurons for desired amount of time. Exchange media every 3-4 days.

Culture cortical neurons for desired amount of time. Exchange media every 3-4 days.

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