- Overview
- Data/Specifications
- Literature/Support
- How To Use
- How It Works
Overview
The NeuroFreeze kit can be used to freeze primary neurons from E18/19 rat and P0/P1mouse hippocampi and cortices. Neurons frozen in this media maintain high levels of viability (up to 90%) upon thawing. These neurons show all the expected characteristics of primary neurons, including appropriate morphology and expression of neuronal and synaptic markers. The availability of this media will enable investigators to freeze primary neurons from embryonic rats (E18) or P0/P1 wild type or genetically engineered mice.
Data/Specifications
Species: mouse, rat
Literature/Support
Product Insert:
References:
Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt LF,Arikkath J. Culturing pyramidal neurons from the early postnatalmouse hippocampus and cortex. Nature Protocols, 7(9):1741-1754(2012).
How To Use
Freezing of Primary Neurons:
- Obtain dissociated primary neurons from rat/mouse hippocampus/cortex as described (Beaudoin et al). Re-suspend dissociated cells in minimal volume of plating media.
- Estimate cell viability on a Hemocytometer.
- Do not exceed a maximum of 4million cells/vial for freezing. It is recommended to freeze 1 to 4 million cells per vial.
- Re-suspend cells to be frozen in 900μl of Reagent A.
- Add 10% (100μlfor 1mL) of Reagent B to the vial.
- Add 1% (10μl for 1mL) of Reagent C to the vial.
- Gently mix and place the vial in isopropanolbath in -80°C freezer.
- Next day, place vial(s) inliquid nitrogen.
THAWING PROTOCOL
- Follow your laboratory’s protocol for coating substrate.
- Add pre‐maintenance media (Reagent D) to dishes/plates and incubate in 37°C incubator for a minimum of 2 hours (recommended 4 hours).
- Bring a vial of cells from liquid nitrogen and quickly thaw in 37°C water bath (usually takes about 90 seconds) – it is critical that thawing is accomplished as fast as possible.
- Estimate cell viability.
- Seed cells onto the substrate (See Plating Density Chart below)
- After cells have attached to the substrate (3-4hours), replace pre-maintenance media with your lab’s neuron maintenance media (B-27 containing maintenance media is recommended).
- Maintain neurons in B27 containing maintenance media at 37°C.
How It Works
Rat Cortical Neurons at DIV14 stained with markers TauI and MAP2
Mouse Cortical Neurons at DIV 21 stained with markers MAP2 and TauI
