- DMEM Medium
- Carbondioxide Chamber
- 70% Isopropyl Alcohol
- Dissection Board
- Surgical Tray
- Iris Scissors
2-ME (2-mercaptoethanol), 50 mM:
Prepare 1 M stock: 0.5 ml 14.3 M 2-ME, 6.6 ml water.
Prepare 50 mM stock: 5 ml 1 M 2-ME, 95 ml water.
Store at 4oC, Dilute to 50 μM (final) for use in media.
Complete DMEM Medium
Dulbecco's modified Eagle medium, high-glucose formulation, containing: 5% FBS, heat-inactivated 1 hr at 56oC, 1% nonessential amino acids, 50μM 2-ME, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate (Pen-Strep solution is available, prepare it to correct dilution).
ACK Lysing Buffer
0.4145 g NH4Cl (0.15 M), 0.05 g KHCO3 (10.0 mM), 1.86 mg Na2EDTA (0.1 mM). Add 30 ml H2O and adjust pH to 7.2-7.4 with 1N HCl . Add H2O to 50 ml. Filter sterilize through a 0.2-μm filter and store at room temperature.
Euthanasia of Mouse: Carbon Dioxide Asphyxiation
- Precharge the impervious container or the CO2 chamber with CO2 prior to introducing the animals.
- Place animals in the container filled with CO2 and replace the lid.
- Unconsciousness will occur within 30 sec, but animals should be left in the container for several minutes to ensure death.
- Verify death by lack of cardiac pulse and fixed and dilated pupils prior to carcass disposal.
Removal of Mouse Lymphoid Organs
- After sacrificing the animal in a humane manner. Place it on its back on clean, dry, absorbent paper [or dissection board].
- Wet the fur with 70% ethanol to sterilize the area and reduce the possibility of contamination.
- Make a midline incision with iris scissors.
- Retract the skin above the head and below the thighs.
Removal of the Spleen
- Make a 1-inch. incision at the left of the peritoneal wall with surgical scissors.
- Grasp as much of the spleen as possible with curved iris forceps.
- Gently pull the spleen free of the peritoneum, tearing the connective tissue behind the spleen.
- Place the spleen in several millilitres of tissue culture medium in a tissue culture plate. [The choice of medium is dependent upon the protocol the cells will be subjected to.]
Removal of the Thymus
- Make an incision in the chest, beginning at the xiphoid and extending to the neck with surgical scissors.
- Retract the ribs with curved forceps. [It may be necessary to crack the ribs for effective retraction. The thymus is a yellowish-white bi-lobed organ found just under the ribs, attached above the heart in the midline.]
- Grasp each lobe of the thymus with curved forceps and gently pull the thymus away. [Because the thymus is delicate, it is not unusual to tear it during removal. Unless maximal cell recovery is required, the tearing will not be harmful]
- Place the thymus tissue in several millilitres of tissue culture medium in a tissue culture plate.
The removed spleen and thymus is placed in a tissue culture medium (complete DMEM medium) and preseved fo further use.
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