Congenic Bone Marrow Transplant Protocol
Using Thy 1.1 or Ly 5.1 donor mice.
Book help for IV injections (Jade C/Mark)
Irradiate recipient mice before collecting donor marrow (or have one person do the irradiation while the other prepares donor BM).
See Jade C before ordering mice to ensure they are housed in the clean area at central animal house. You need to shower in to access both the mice and the irradiator room.Mice can then be moved to a less clean environment if required.
Irradiate Mice - Lethal dose used is usually 2 x 550 RADs
Irradiation Doses should be 2-3 hours apart (to allow for mobilization of resting poietic cells after the initial irradiation)
After second dose wait 1 hour before IV injection of Donor BM.
The irradiator uses a large circular canister capable of holding many mice at once
It is possible to make two seperators out of cardboard to divide it into four sections (can fit 5 mice in each section thus irradiating 20 mice at a time). This cuts down the time required for irradiations by three quarters. Proper seperators may be being organised? Otherwise just ask me (Marc)
Gold standard BM dose (For post irradiation reconstitution) is 5 x 10^6 cells per mouse
Eg: bone marrow suspension contains 2.054 x 10^8 cells and 40 doses of BM are required (ie. 30 recipients + 10 extra doses to be careful)
(5x10^6 / 2.054x10^8) x 1000 = 24.34uL (amount of 1mL suspension containing 5x10 6 cells)
We want to inject 100uL into each mouse containing 5x10^6 cells
100uL – 24.34uL = 75.66uL (RPMI to be added per dose)
So for 40 doses:
24.34uL x 40 = 973.6uL (Amount of initial 1mL cell suspension required)
75.66uL x 40 = 3026.4uL (Amount of RPMI to be added to make up 40 doses)
Give mice seeds , they've had a rough day.