Tissue culture general procedures

Methods General tissue culture procedures are followed as previously described (34). Tumor cells and HUVECs are maintained in culture under standard conditions (37 °C, 5% CO, 95% humidity) and passaged when subconfluent. 

An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. 

Uhrathin sectioning is used to prepare specimens that are too large to view di-recdy with the electron beam, e.g., animal and plant tissues, fungi, bacteria and cultured cells, and organelle preparations. It is the basis for most of the im-munolabelling and in situ hybridization techniques. Both the theory and practice of uhrathin sectioning have been well described in a number of texts and laboratory manuals (28-31), so it is sufficient here to describe only the general procedure as a basis for the special applications for biochemical research. 

Tissue culture techniques generally require very exacting media with numerous components and are often plagued by bacterial contamination. A recent interest in the use of cultured hepatocytes as models for metabolism offers some advantages, but still requires a tedious procedure of perfusion of a section of liver and seeding of isolated hepatocytes in a complex nutrient medium, followed by daily maintenance of the cultures [25]. 

General ISH procedures are given in Table 11.4. Sectioned tissues or cultured cells are used and probes are detected, after hybridization and washes, by autoradiography or nonradioactive methods. 

---