Preparation of lymphocytes from solid lymphoid organs
6.3 Preparation of lymphocytes from solid lymphoid organs
Lymphocyte suspensions may be prepared from solid lymphoid organs, for example spleen, lymph nodes, bursa, thymus.
METHOD
1 Tease the organs apart using forceps or needles over fine nylon gauze to retain the connective tissue capsule of the organ.
2 Wet the gauze with a few ml of tissue culture medium. Lymphocyte viability tends to vary with the amount of fibrous tissue in the organ and the ‘deftness of touch’ of the operator.
Approximate cell yields from each of the organs are given in Fig. 6.2. Cell viability should be as follows: thymus 95%, spleen 80–90% and lymph node 70–80%. The lymphocyte subpopulations of each organ are summarized in Table 6.1. If uniformly viable cell suspensions are required, e.g. for antibody-mediated cytotoxicity assays, dead cells may be removed as described in Section 6.4.
Table 6.1 Percentage lymphocyte subpopulations of murine lymphoid organs Organ
% T lymphocytes
% B lymphocytes
% ‘null’ cells
Thymus
97 1 2 Lymph node
77 18 5 Spleen
35 38 27 Blood
70 24 6 Thoracic duct lymph
80 19 1 T lymphocyte: thymus-derived small lymphocyte, with Thy-1-positive, surface immunoglobulin-negative phenotype.
B lymphocyte: in chickens B lymphocytes are bursa-derived (i.e. from the bursa of Fabricius); in mammals, B lymphocytes may be defined serologically by their expression of surface immunoglobulin but not Thy-1 antigens.
‘Null’ cells: resemble small lymphocytes morphologically, but do not have Thy-1 or surface immunoglobulin surface markers. This is a mixed cell lineage, some members of which are undoubtedly of the lymphoid series and, having IgG Fc receptors, are capable of antibody-dependent cytotoxic activities. These cells have some of the characteristics of monocytes and express some T-lymphocyte markers. Typically, these have the morphology of large granular lymphocytes (Fig. 7.2c). Natural killer (NK) cells also belong to this population.
6.3PREPARATION OF LYMPHOCYTES FROM SOLID LYMPHOID ORGANS
Axillary vessels
These are the vessels
Cervical lymph
severed for underarm bleeding node
Axillary lymph node
In or adherent to the musculature of the arm
Thymus
Primary lymphoid organ.
Mesenteric lymph nodes
Lymphocyte Three large unpaired nodes
content 1–3 × 10 8 suspended in mesentery supporting the intestine. Lymphocyte content
2–3 × 10 7 (all other lymph nodes yield about 1–2 × 10 7 lymphocytes)
Spleen
Major peripheral lymphocyte organ. Lymphocyte content 1–2 × 10 8
Peyer's patches
White raised areas on
Epigastric or inguinal
the side of the intestine
lymph node
away from the mesentery Frequently these paired nodes are asymmetrically enlarged suggesting some antigenic challenge, perhaps an infection, in the area drained by these nodes
Femur
Usual source of bone marrow. Yields about 1–2 × 10 7 cells
Fig. 6.2 The major lymphoid organs of the mouse. One of the major lymphoid organs not obvious in
the drawing is the blood; it contains about 3–10 × 10 6 lymphocytes/ml in a total leucocyte count of 4–12 × 10 6 /ml. As a guide, the differential leucocyte count of mice is approximately neutrophils 25%, eosinophils 2%, basophils < 0.1%, lymphocytes 65% and monocytes 8%. The data for lymphocyte content of the various organs were derived from a specific pathogen-free (SPF) stock of CBA mice aged about 4–8 weeks old. At this age the total blood volume is about 6.3 ml/100 g body weight. The change in blood volume with weight (age) shows a curvilinear relationship probably due to an enhanced fat deposition after 20 g. It follows the general form y = 0.097x − 0.002x 2 where y is blood volume (ml) and x is body weight (g). Other strains of mice, particularly if they are outbred, show a linear relationship between body weight and blood volume. For example, the relationship for Swiss mice is: y = 0.072x where unknowns are as above. The cell content of each lymphoid organ is known to vary with age (especially the thymus), strain, sex, health and immune status of the individual.