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 spleen. Thaw enough to allow slicing into 4-5g
chunks and transfer to homogenisation container.
- Homogenise approx. 20g and 180 mL sucrose solution in sealed
250 mLcontainer using programmable Omni-Mixer. Setting
at 4000rpm, 6 x 30seconds.*
- Transfer homogenate to 2L beaker on ice/salt. Stir on magnetic
stirrer.
- Repeat homogenisation : 4 x 20g spleen to create a pool of
1L of 10%-spleen homogenate.
- 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 5 minutes.
- Transfer onto dry ice for transport to storage facility
at NIBSC (-80 oC).
* Shown to be adequate by facsimile homogenisation of frozen
human spleen