Flash link:  https://vlab.amrita.edu/repo/BIOTECH/IMM/Removal_Thymus_and_Spleen_from_Mouse/index.swf

 

Materials Required

 

• DMEM Medium
• Carbondioxide Chamber
• 70% Isopropyl Alcohol
• Dissection Board
• Surgical Tray
• Iris Scissors
• Petridishes
• Forceps

 

Regent Preparation

 

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.

 

Procedure

 

Euthanasia of Mouse: Carbon Dioxide Asphyxiation

 

1. Precharge the impervious container or the CO2 chamber with CO2 prior to introducing the animals.
2. Place animals in the container filled with CO2 and replace the lid.
3. Unconsciousness will occur within 30 sec, but animals should be left in the container for several minutes to ensure death.
4. Verify death by lack of cardiac pulse and fixed and dilated pupils prior to carcass disposal.

 

Removal of Mouse Lymphoid Organs

 

1. After sacrificing the animal in a humane manner. Place it on its back on clean, dry, absorbent paper [or dissection board].
2. Wet the fur with 70% ethanol to sterilize the area and reduce the possibility of contamination.
3. Make a midline incision with iris scissors.
4. Retract the skin above the head and below the thighs.

 

Removal of the Spleen

 

1. Make a 1-inch. incision at the left of the peritoneal wall with surgical scissors.
2. Grasp as much of the spleen as possible with curved iris forceps.
3. Gently pull the spleen free of the peritoneum, tearing the connective tissue behind the spleen.
4. 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

 

1. Make an incision in the chest, beginning at the xiphoid and extending to the neck with surgical scissors.
2. 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.]
3. 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]
4. Place the thymus tissue in several millilitres of tissue culture medium in a tissue culture plate.

 

Result

 The removed spleen and thymus is placed in a tissue culture medium (complete DMEM medium) and preseved fo further use.

 

 

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