Simplified Protocol for Plating and Culturing Primary Neurons (Hippocampal and Cortical)
Time required: 2 - 4 hours
Preparation time: 1 day prior to use
MEM/FGH Medium - plating medium: MEM with 10% FBS, glucose and HEPES
NB27G - maintenance medium: Neurobasal with B27 and GlutaMAX
(aliquots of MEM/FGH and NB27G medium to be used with cells must be temperature/pH prebalanced
by placing in a tissue culture dish for at least 1hr in the incubator)
PL Solution - coating solution (PL): poly-L-Lysine 0.1 mg/ml in borate buffer
Coating (dishes or coverslips)
If you are using glass bottomed culture chambers, regular multi-well plate or cell culture dishes,coat them with PL solution overnight.
The next morning, wash with sterile water 3X and fill with MEM/FGH medium, and balance in CO2 incubator for at least 2 hours before plating.
If you are using coverslips for culturing, first place coverslips in 70% nitric acid overnight, wash 8-10X with excess volume (at least 10X volume of acid added) milliQ purified water, and bake them in oven at 225degC for 4 hours or longer (alternatively, if using coverslip holding racks, UV sterilize in tissue culture hood with blower on for several hours).
Coat coverslips as described in steps 1 & 2.
Thaw
Prebalance several mL of MEM/FGH medium in CO2 incubator for 30 minutes prior to thawing cells in tissue culture dish.
If you are planning on thawing multiple vials, perform the following steps one vial at a time.
Take a vial, put it in 37degC water bath and take it out when almost all ice is thawed, but do not keep the vial in the water bath for more than 2 minutes.
Spray the outside of the vial with 70% ethanol and wipe with paper towel. Quickly proceed to the plating step for each vial.
Plate*
*Note - See plating chart below for guidelines for optimal neuron morphology. Plating neurons at higher density will yield more robust cultures over time. To ensure healthy cultures, neurons should not be plated at a density lower than 300K per 60mm dish, 100K per well of 6-well dish or 35mm dish, or 50K per 12-well dish.
Slowly pipette the thawed cell solution with 1ml tip to pre-coated plates/dishes/coverslips (depending on your application and cell density), avoiding any rapid pipetting and tips with small openings (wide bore at least 1.5mm). Use plating chart below for guidelines.
Take 1 ml of MEM/FGH medium and place back in vial to wash any neurons that remain at the bottom and on tube walls. Pipette out the 1mL to the plates/dishes/coverslips equally.
Swirl the plates/dishes/coverslips slowly for 20 seconds by hand rotation, in opposing directions, to equally distribute the neurons and return to CO2 incubator.
Repeat thaw and plate steps for all vials.
Neurons should start attaching within 30 minutes after plating. After 2-4 hours, replace MEM/FGH medium with pre-balanced NB27G medium.
After 3-4 days, remove 1/3 of old NB27G medium from neurons and replace with an equal volume of fresh, pre-balanced (in CO2 incubator) NB27G medium (can add 1.25uM cytosine arabinose final concentration of total medium; use stock 3.75uM in NB27G). Repeat this procedure on day 7, 14 and 21, without cytosine arabinose addition.
MEM/FGH Medium – MEM (with Earles salts and sodium pyruvate, no glutamine) with 10% FBS, 33mM glucose and 10mM HEPES. Adjust pH to 7.3 and sterile filter.
NB27G - maintainence medium: Neurobasal (Life Technologies) with B27 (Life Technologies) and GlutaMAX (Life Technologies). Okay to substitute B27 with GEM21 NeuroPlex from Gemini Bio Products.
PL solution: Make up 0.1mg/mL poly-L- or poly-D-lysine from 1.0mg/mL stock dissolved in borate buffer (dissolve 0.72g borax (sodium tetraborate) and 0.81g boric acid in 100ml of water, pH to 8.5) and sterile filter. This must be made up fresh, and plates not allowed to dry after PL solution coating.
Quality Control
AccelBio offers research on primary cells of a higher functional and morphological quality than most freshly isolated cells used currently in laboratories. They are routinely tested for sterility and mycoplasma toxins for each lot.
Our neurons have been screened for synapse number, dendrite and axon morphological markers, intrinsic excitability, and gene expression profiling, and consistently show no significant differences from freshly isolated cells. Data packets are available upon request.
Viability
Our cryopreserved cells always display the highest viability, over 85% after thawing. For neurons, this results in a much greater number of healthy and morphologically superior cell cultures after plating for weeks in vitro.
We are setting the industry standard for performance and maximization of the time scientists spend working on living cells.
Strains
We provide users with strain data upon request. Our standard orders of hippocampal and cortical neurons are derived from wild-type C57BL6 or Swiss mice, as well as Sprague-Dawley rats.
We are currently taking custom requests for specific animal strains needed for any cell type desired, including many genetically modified animals. Contact our scientific operations for details.
Genomic profiling (by microarray / GWAS) - no significant difference
Dendritic branching (by Scholl analysis) - no significant difference
Synapse numbers and density (using classic markers such as synapsin) - no significant difference in dendrites or cell body
Neurite Outgrowth (by time-lapse microscopy) - no significant difference
Excitatory and Inhibitory Synapse Development (using vGLUT and GAD65) - no significant difference
Instrinsic Excitability (measuring field potentials, EPSPs and IPSPs) - no significant difference
