Equipment :
Eppendorf EDOS 5222 Liquid dipensing system
CamLab Omni-Mixer ES Homogeniser
Class 2 Microbiological Safety Cabinet
Solution :
0.25M-sucrose (MW 342.30) : 85.56g/L in pyrogen-free water.
Pre-chilled to ~4 degrees centigrade
Procedure:
- Weigh frozen brain regions. Thaw to allow slicing into 4-5g
chunks and transfer to homogenisation container.
- Homogenise to yield 10% (w/v) homogenate solution in sealed 250
mL container using programmable Omni-Mixer. Note due to weight of
obex a 5% (w/v) homogenate was prepared. Setting at 4000rpm, 6 x
30seconds.*
- Place homogenate bottle on ice until ready for dispensing.
- Wash homogenisation bit by running machine (washing step in
done between homogenisation of different regions of the brain);
Setting at 4000rpm, 6 x 30seconds with sucrose solution.
- Dispense 100 x 0.5 mL into pre-labelled, Simport 1.2mL
cryovials, immerse in bleach (10%-Chloros) for >15 minutes at
4oC.
- Rinse in water to remove surface bleach. "Flash Freeze " by
immersing in liquid nitrogen for 10 minutes.
- Transfer onto dry ice for transport to storage facility at
NIBSC (-80 oC).
* Shown to be adequate by facsimile homogenisation of frozen
sheep brain