2. Euthanize mice as per your IACUC protocol. We utilize CO 2
asphyxiation followed by cervical dislocation as secondary
method of euthanasia.
3. Inside the biosafety hood, spray the mouse with ethanol and
cut open the abdomen just below the rib cage to snip the portal
vein and collect the blood in Microtainer tubes (as explained in
Subheading 3.4).
4. Make an incision of the skin in the mid-abdomen and remove
the skin from the distal part of the mouse including the skin
covering the lower limbs.
5. Cut off the muscles from the lower extremities using scissors
and carefully cut at the hip joint, while avoiding breaking the
femur head.
6. Remove the remaining muscles from the femur and tibia using
a scalpel and scissors and separate the femur from the tibia at
the knee joint. Be careful not break the bone ends. Place the
bones in a petri dish containing ice-cold RP-10.
From now till step 15 all procedure must be done on ice/4
C.
7. In a petri dish, quickly rinse each bone with ice-cold 70%
ethanol followed by three washes in ice-cold sterile PBS to
wash off the ethanol.
8. Inside a new petri dish, snip off the epiphyses at both ends of
the bones. Remove more tissue until it becomes easy to see
bones. Make sure enough is cut off from the tip to allow
insertion of a needle for flushing out the marrow.
9. Using a 25-G needle and a 10 ml syringe prefilled with RP-10
and 2 mM EDTA, flush the bone marrow cells from both ends
of the bone shafts into a 50 ml screw top sterile Falcon tube.
Use 2 ml for each side until bones look clear and not reddish
(see Note 12).
10. Use pipette to gently mix the cells and disrupt clumps.
11. Filter the cell suspension through a 100 μm filter strainer into
a 50 ml tube placed on ice.
12. Centrifuge at 328 rcf for 5 min at 4
C.
13. Lyse the red blood cells by resuspending the cell pellet in 5 ml
of Lysis Buffer and mixing up and down gently using a
pipette. Wait 1–2 min on ice and then neutralize with 5 ml
of HBSSÀ (see Note 13).
14. Centrifuge for 5 min at 328 rcf at 4
C. Aspirate out the
supernatant and resuspend the pellet in 5 ml of RPMI supplemented with 10% FBS and 2 mM EDTA.
15. Centrifuge again for 5 min at 328 rcf at 4
C.
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Manmeet Bhalla et al.
asphyxiation followed by cervical dislocation as secondary
method of euthanasia.
3. Inside the biosafety hood, spray the mouse with ethanol and
cut open the abdomen just below the rib cage to snip the portal
vein and collect the blood in Microtainer tubes (as explained in
Subheading 3.4).
4. Make an incision of the skin in the mid-abdomen and remove
the skin from the distal part of the mouse including the skin
covering the lower limbs.
5. Cut off the muscles from the lower extremities using scissors
and carefully cut at the hip joint, while avoiding breaking the
femur head.
6. Remove the remaining muscles from the femur and tibia using
a scalpel and scissors and separate the femur from the tibia at
the knee joint. Be careful not break the bone ends. Place the
bones in a petri dish containing ice-cold RP-10.
From now till step 15 all procedure must be done on ice/4
C.
7. In a petri dish, quickly rinse each bone with ice-cold 70%
ethanol followed by three washes in ice-cold sterile PBS to
wash off the ethanol.
8. Inside a new petri dish, snip off the epiphyses at both ends of
the bones. Remove more tissue until it becomes easy to see
bones. Make sure enough is cut off from the tip to allow
insertion of a needle for flushing out the marrow.
9. Using a 25-G needle and a 10 ml syringe prefilled with RP-10
and 2 mM EDTA, flush the bone marrow cells from both ends
of the bone shafts into a 50 ml screw top sterile Falcon tube.
Use 2 ml for each side until bones look clear and not reddish
(see Note 12).
10. Use pipette to gently mix the cells and disrupt clumps.
11. Filter the cell suspension through a 100 μm filter strainer into
a 50 ml tube placed on ice.
12. Centrifuge at 328 rcf for 5 min at 4
C.
13. Lyse the red blood cells by resuspending the cell pellet in 5 ml
of Lysis Buffer and mixing up and down gently using a
pipette. Wait 1–2 min on ice and then neutralize with 5 ml
of HBSSÀ (see Note 13).
14. Centrifuge for 5 min at 328 rcf at 4
C. Aspirate out the
supernatant and resuspend the pellet in 5 ml of RPMI supplemented with 10% FBS and 2 mM EDTA.
15. Centrifuge again for 5 min at 328 rcf at 4
C.
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Manmeet Bhalla et al.