Preparation of lymphocytes

Carry out all procedures under strict aseptic conditions. Collect blood into anticoagulant and process immediately or store at ambient temperature (not refrigerated) for up to two days. 

Layer blood carefully over Ficoll-Triosil in centrifuge tubes, using a ratio of two volumes of blood to one volume density gradient medium. 

Collect a sample of plasma and store frozen to act as a control for the desired antibody. 

Using a pipette, collect the layer of white lymphocytes from the interfece between the plasma and Ficoll. and place in a fresh centrifuge tube. Some medium and plasma will be collected as well, but ensure that the tube is less than half filled with cell suspension. 

Fill the tubes with Hep-RPMl and centrifuge at 800 g for 5 min to pellet the cells. 

---

The amounts of proteins adsorbed on the surface of glassware has a significant effect on the quantitative analysis of very small amounts of protein in the case of radioimmunoassay or enzymeimmuno-assay (3 ). Columns of glass beads with bound antigens were used for the preparation of lymphocytes to prove the clonal selection theory (3 ). Flagellin of Salmonella was found to be adsorbed on glass surfaces (3 ). Boone (3 ) and Kataoka (3 ) separated... 

Preparation of lymphocytes for analysis of intracellular cytokines in vitro... 

Small scale preparation of mononuclear cells (MNQ1 FicoU-Hypaque gradient centrifugation 385 Preparation of lymphocytes ftir analysis of intracellular cytokines in vitro 386 Staining for intraceUnlar cytokines in activated mononuclear cells in vitro 387 Preparation of monotytes for measnrement of intracellular cytokines in vitro 388... 

The basic principle underlying the LLNA is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the site of chemical application. This proliferation is proportional to the dose applied and to the potency of the allergen, and provides a measurement of sensitization. The LLNA assesses this proliferation as a dose-response in which the proliferation in test groups is compared to that in vehicle treated controls. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index , is determined, and must be at least three before a test substance can be further evaluated as a potential skin sensitizer. The test substance, plus vehicle and positive control, is applied for three consecutive days to the ears of test mice, on days 4 and 5 the animals are left alone, and on the 6th day they are prepared for the proliferation assay, sacrificed, and the measurements are done. 

Preparation of T-lymphocyte lines and clones with specificities to preselected protein sitesT... 

Anti-lymphocyte globulin (ALG) has been prepared as an highly purified solution of y-globulins with antilymphocyte activity by immunizing horses with human lymphocytes. It activates complement-mediated destruction of lymphocytes and thus decreases cellular immunity with only a limited effect on humoral immunity. Anti-lymphocyte globulin suppresses delayed type hypersensitivity reactions. It is used for the prevention and treatment of rejection episodes of transplanted organs. It also has some indication for the management of idiopathic aplastic anemia. Adverse effects include pain at the site of injection, erythema, serum sickness and rarely anaphylactic shock and thrombocytopenia. 

The preparation of any MAB commences with the generation of lymphocytes sensitized to the antigen of choice. Conventionally this is done by the immunization of a mouse against the antigen followed by repeated booster injections until a suitable serum response has been obtained. Optimal results are obtained if the immune... 

Apart from the spleen, other lymphoid tissues, such as tonsils and the mesenteric or popliteal lymph nodes, can be used as a source of lymphocytes. In the preparation of MABs of human or veterinary origins it is often not possible to obtain lymphoid tissue, and there have been many reports of the successful use of lymphocytes separated from peripheral blood. In some cases, for ethical or practical reasons, it is not possible to immunize the lymphocyte donor, as when human MABs are required, or acutely toxic antigens are used. Also, antigen is not always available in sufficient quantities to perform a successful immunization in vivo. In these circumstances, it may be possible to perform the boosting stage or, indeed, the entire immunization procedure on the lymphocytes in vitro. 

To circumvent some of the limitations of direct immunization, phage display technology has been applied to the preparation of fully human MABs. Gene libraries of cDNA from nonimmune or immunized donor lymphocytes are expressed in bacteriophages. The bacteriophages display functional antibody fragments and can... 

Fig. 6.12. Back-gating from CD14/CD45 fluorescence to determine the scatter characteristics of lymphocytes. Such back-gating facilitates the placing and then evaluation of a lymphocyte scatter gate within a peripheral blood mononuclear cell preparation.

The problem is that if an individual antibody-producing cell is isolated and grown in culture, its descendants have a limited lifespan that severely limits their use for the routine preparation of monoclonal antibodies. In 1975, Milstein and Kohler discovered how monoclonal antibodies of almost any desired antigen specificity can be produced indefinitely and in large quantities. Their method was to fuse a B lymphocyte producing antibody of the desired specificity with a cell derived from a cancerous lymphocyte tumor, called a myeloma cell, which is immortal. The cell fusion is called a hybridoma, which is both immortal and secretes the same specific antibody originally encoded by the B lymphocyte. 

Cell sorting takes place at a rate of 300,000 cells/min and for this reason a FACS machine is more usually used as an analytical tool rather than in a preparative mode. In addition to its use in cell cycle analysis, it can be used (a) to analyse the distribution of lymphocytes carrying a series of different surface antigens (e.g. to determine the proportion of T4 lymphocytes in a blood sample) (b) to estimate the proportion of dead cells in a population (i.e. cells which stain with propidium iodide without prior fixation) or (c) to determine the proportion of transformed cells (i.e. cells bearing a particular surface antigen) in a culture or biopsy (Watson, 1987). 

---