Congenic Bone Marrow Transplant Protocol

Contents:

 

 

 

 

Preface

Using Thy 1.1 or Ly 5.1 donor mice.

Donor Numbers:

  • Approx 1:4 Donor/Recipient Ratio
  • Work on the assumption of getting a minimum of 2 x 10^7 BM cells per mouse

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.

Recipient Preparations

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)

I - Donor BM Preparation

  • Kill Ly5.1 Mice (Ly5.1/5.2 Donor/Recipient difference)
  • Remove leg bones (Tibia and Femur)
  • Remove ends of bones with dissecting scissors (preferably old blunt scissors, or use the lower part of the scissors so as not to blunt the sharp tips).
  • Flush the marrow from the bones into a sterile petri dish with sterile RPMI using a 26gauge needle and a 3ml syringe.
  • Suspend the bone marrow cells by drawing the clumps (“worms”) back through the needle of the syringe, and expelling back into the petri dish. (Use a 22-23g needle to reduce shearing forces on cells or a blue tip/pipette)
  • Rinse the syringe once through with clean RPMI to collect any cells left inside.
  • Transfer the cells/RPMI suspension into a 10 or 50 ml tube through a sterile filter.
  • Spin at 1500 for 5 mins at 4°C.
  • Remove supernatant.
  • Resuspend in 1mL RBC Lysis Buffer for 1 minute (use more Lysis buffer if pooling BM from multiple donors ie. 3-5mL depending on how much BM)
  • Dilute with RPMI (approximately 7-10times dilution will inactivate lysis buffer)
  • Spin at 1500 for 5 mins at 4°C.
  • Remove supernatant.
  • Resuspend cells in a known volume of sterile RPMI
  • Count Cells using Coulter Counter or Hemocytometer (Obtain in Cells/mL)

II - The Math Stuff

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)

III - Donor BM Preparation

  • Adjust cell suspension so one dose (e.g. 5 x 10^6 cells) is contained in 100μl
  • Transfer cells to Eppendorf tubes and take a box of 1mL insulin needles to Central Animal House for I.V. injections.
  • Cells tend to come to rest at the bottom of the tube so shake before filling needles
  • Best to take up 3-4 doses in a needle at a time (to minimise clumping induced blockages in the needle)

Give mice seeds , they've had a rough day.