"Removal of Lymphoid Organs". In: Current Protocols in ... - IMBA

SELECTED SURGICAL PROCEDURES
Removal of Lymphoid Organs
The removal of lymphoid organs for further culture, isolation of cell populations, or direct
testing is one of the simplest and most frequently used procedures in immunology. Typical
uses include providing cells for cytotoxicity or proliferation assays, for analysis of
cytokine production or response, and for the isolation of B, T, and adherent cell subpopulations
for culture or reconstitution of other animals. This unit covers the identification
and removal of mouse lymphoid organs, but the same protocols are applicable to other
rodents and rabbits with minor modifications.
REMOVAL OF MOUSE LYMPHOID ORGANS
Materials
70% ethanol
Hanks balanced salt solution (HBSS; APPENDIX 2) or tissue culture medium
Iris scissors (straight or angled)
Iris forceps (curved)
Tissue culture plate
Additional reagents and equipment for euthanasia (UNIT 1.8)
Prepare the animal
1. Sacrifice the animal in a humane manner. Place it on its back on clean, dry, absorbent
paper.
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 (Fig. 1.9.1).
4. Retract the skin above the head and below the thighs by pulling it with gloved fingers.
5. Turn the animal to expose its left side for spleen (steps 6 to 9) and lymph node (steps
10 to 12) removal. Keep the animal on its back for thymus (steps 13 to 16) removal.
Remove the spleen
6. Make a 1-in. incision at the left of the peritoneal wall with surgical scissors.
The spleen is attached to the greater curvature of the stomach by connective tissue.
7. Grasp as much of the spleen as possible with curved iris forceps.
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Contributed by J.P. Reeves and P.A. Reeves
Current Protocols in Immunology (1991) 1.9.1-1.9.3
Copyright © 1991 by John Wiley & Sons, Inc.
SECTION III
UNIT 1.9
BASIC
PROTOCOL
Care and
Handling of
laboratory
Animals
1.9.1
Supplement 1

Removal of
Lymphoid Organs
1.9.2
8. Gently pull the spleen free of the peritoneum, tearing the connective tissue behind
the spleen.
Alternatively, the top and bottom portions of the spleen can be grasped and torn from the
connective tissue one part at a time. It is usually more efficient to remove the spleen all at
once, particularly if large numbers of animals will be used.
9. Place the spleen in several milliliters of HBSS or tissue culture medium in a tissue
culture plate.
The choice of medium is dependent upon the protocol the cells will be subjected to. See
Chapters 3 & 4.
Remove the lymph nodes
10. Identify the lymph nodes as shown in Figure 1.9.2.
The major lymph nodes are found in the axillary, cervical, inguinal, and mesenteric regions.
In normal, nonimmunized animals maintained in clean facilities, the lymph nodes are
usually quite small and can easily be overlooked. Axillary nodes can be readily identified
upon dissection in this region, and occasionally the adjacent brachial nodes are found in
the triceps area. Cervical nodes flank the trachea and are beneath the large salivary gland.
Inguinal nodes are found in the groin area in a fatpad attached to the skin, but after
retraction of the skin, are often found buried in fat at or near the knees. Mesenteric lymph
nodes lie deep in the intestinal mesentery. Both cervical and mesenteric nodes are
frequently overlooked. Typically, 1-5 × 10 7 lymph node cells can be isolated from a single
mouse.
11. Grasp the lymph nodes with curved forceps and pull them free of attached tissue.
Occasionally, it is necessary to dissect adjacent fat.
12. Place the lymph nodes in several milliliters of HBSS or tissue culture medium in a
tissue culture plate.
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Supplement 1 Current Protocols in Immunology

Remove the thymus
13. Make an incision in the chest, beginning at the xiphoid and extending to the neck
with surgical scissors.
14. 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.
15. 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. Thymic size varies with age, being
maximal at 3 to 5 weeks in the mouse and shrinking to a minimum at ∼6 months. The number
of cells obtained is related to the age of the animal and the size of the thymus.
16. Place the thymus tissue in several milliliters of balanced salt solution or tissue culture
medium in a tissue culture plate.
Key References
Sjodin, K., Dalmasso, A.P., Smith, J.M., and Martinez, C. 1963. Thymectomy in newborn and adult mice.
Transplantation 1:521-525.
Van Alten, P.J. 1984. Thymectomy and engraftment of thymic tissue. Methods Enzymol. 108:10-19.
Contributed by J.P. Reeves and P.A. Reeves
Food and Drug Administration
Bethesda, Maryland
Care and
Handling of
laboratory
Animals
1.9.3
Current Protocols in Immunology Supplement 2