AUTHOR: Michelle Sandau

Date last revised: 7/25/05

Standard Operating Procedure

Irradiation Bone Marrow Chimeras

Hazards:

EXPOSURE TO ANIMALS: Personnel involved with animals may be exposed to bites, scratches, infectious agents, and allergens.

TETANOUS: a rare but potentially fatal bacterial infection is possible after any animal bite.

See also the Boynton Health Service brochures on animal bites and allergies in this manual.

CARBON DIOXIDE: toxic gas, avoid inhaling.

See also: General Tissue Culture S.O.P. in this manual.

Protection:

Always wear gloves and lab coat when handling animals or animal carcasses. Wear a face mask if you’re allergic to mice or to prevent the onset of allergies.

Have current tetanus immunization.

If biten by a mouse, immediately wash wound with soap and water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).

Waste:

Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 2nd floor of Nils Hasselmo Hall.

Spill Clean-Up:

Any spills should be placed in the autoclavable biological waste bags.

Procedure:

Pretreatment of hosts (day –2)

Treat mice with antibiotic water from day –2 onward (until > 1 month post-irradiation)

Mice which express different class I MHC molecules compared to the bone marrow donors (e.g. H-2d -> H-2bxd or H-2b TAP- -> H-2b) will need to be treated with anti-NK antibodies.

Inject with 200mg of PK136 (anti-NK1.1) i.p. on days –2 and –1 relative to irradiation

Irradiation of host (day 0)

Place hosts in wheel

Turn the power on irradiator, open the door, put in attenuator, start turning, close, set to 12.60 min (1000rads) + RESET!, release source, turn on. OR irradiate 6.30 min and repeat 4 hours later.

Return hosts to their cages

BM preparation

**** antibody concentrations will vary between batches, all antibodies should have record of being tested, use at that suggested concentration****

Reagents: media: HBSS+1%FCS in syringe on ice (about 10ml/mouse)

RP10

RPSC

ACK lysing buffer

Low tox. rabbit complement (@-20)

30H12 (Thy1.2) ascites: 1mg/ml (@-70, 2-1-C) or Supe added directly to cells at 2x107cells/mL supe.

IA14 (anti-Thy1.1) 1.2mg/ml (-70, 2-1-B)

2.43 (anti-CD8) Ab (@-70, 2-1-B): 1.1mg/ml - brown cap

Sacrifice mice, skin legs and cut as much muscle as possible, use kimwipe to remove sticky muscle.

Collect femur, tibia and humerus in petri dishes with media.

Squirt bone morrow out of bone with 27 gauge needle(move up and down + from both sides ->bone clears), into 15 ml conical or 50 ml tube depending on number of samples.

Filter suspended marrow through nitex filter and wash at 1000rpm for 5 min in media

Resuspend and count the cells after adding 90ml of ACK + 10ml of cell suspension – take 10ml out of this and add Trypan blue – recovery between 1-4x107/ mouse is typical depending on age of mice. Note: Older mice are the best donors.

Counts:

T cell depletion of bone marrow

**** antibody concentrations will vary between batches, all antibodies should have record of being tested, use at that suggested concentration****

Resuspend bone marrow suspension to about 2x107/ml in 30H12 supernatant directly to cell pellet. If purified 30H12 is available: resuspend cells at 2x107 cells/mL HBSS/FCS + 1mg/ml of 30H12 or 1ml of IA14 ascites. Incubate 30 min on ice.

Wash 2x in RPSC.

Add 14ml of RPSC + complement (1 vial of complement resuspended in 1ml of ice cold ddH2O)

Resuspend cells at 1x107/ML in RPSC + complement

Incubate 30min @ 37

Wash cells in RP10 spin @1000rpm for 5 min. Repeat.

Count cells.

Bystander cell loss is variable, but can be up to 50% after complement treatment. Be gentle when resuspending cell pellets, especially after washes in serum free media because cells are more likely to die due to shearing.

Reconstitution

Inject 5-10x106 depleted BM cells in 200ml of sterile PBS i.v. through tail vein of irradiated recipient mice.

2. Keep on ATB water for > 1 month after reconstitution

3. Wait one month before testing mice for chimerism

Anti CD8 treatment (day 1) -if necessary:

Use 2mg/ 200ml sterile PBS/ mouse of 2.43 Ab (c: 1.1mg/ml)

Inject i.p.

3. Repeat injections (day 2)