M.Fero — May 5, 2003
4 mice from each genotype
4 Ly5 mice
Buckets with wet ice 3x
Bucket with dry ice 1x
Dewar flask with liquid nitrogen
100 mL beakers with 95% ethanol 2x
100 mL beaker with dd. H2O 2x
Sterile petri dishes
Sterile IMDM medium aliquoted into 50 mL conical tubes
Sterile Ficoll-Hypaque (d = 1.077)
Halothane in a bell jar
RBC lysis buffer
- Weigh mouse.
- Anesthetize with halothane or isoflourane inhalation in a bell jar.
- Place the ends of surgical instruments in a 100 mL beaker with ethanol. Keep a second beaker with sterile water or PBS for washing the instruments. Wet the skin with 70% ethanol. Open the skin and pin back to keep the body wall sterile.
- Open the chest and aspirate blood from the heart into a 1 mL syringe through a 23G needle. Transfer blood to Eppendorf tube with a drop of EDTA. Mix continually to prevent clotting. Spot 10 µL onto the end of a glass slide and make a thin smear. Stain the smears with HemaStain according to the standard protocol. Perform RBC and WBC counts on hemacytometer. Perform differential counts of 200 WBC on the blood smears.
- Remove and weigh thymus. Place into 1.5 mL eppendorf tube and plunge into liquid nitrogen. Store on dry ice and then freeze at -80C.
- Remove spleen and place it in a sterile petri dish, record weight, keep dish on ice.
- Remove femurs taking care to trim off as much muscle as possible. Keep instruments clean by washing in water or PBS and dipping ends into 95% EtOH.
In cell culture hood:
- Cut ends off of femurs and flush 1 mL of IMDM through each end into a 15 mL conical tube.
- Mince spleen with two sterile scalpels in a few mL of IMDM. Transfer suspended cells to 15 mL conical tube.
- Pool bone marrow from each genotype and load onto Ficoll in two 15 mL conical tubes. Do the same with spleen cells.
- Spin Ficol gradient at 2500 RPM at 4 deg. C for 15 minutes.
- Remove buffy coat by gently pipetting and transfer it to a fresh conical tube. Q.S. to 15 mL with IMDM. Add 10 µL to 10 µL trypan blue for cell counts. Pellet the cells at 1500 RPM. Carefully aspirate supernatant. Resupend pellet by flicking tube and resuspend in IMDM. Adjust volumes to 1 million cells/mL.