Robert I. Mishell

Immunization of Normal Mouse Spleen Cell Suspensions in vitro

Dissociated cells from the spleens of unimmunized mice were cultured with and without various mammalian erythrocytes. Spleen cell suspensions cultured with heterologous red cells developed levels of hemolytic plaque-forming cells only one log2 less than those seen in vivo. The reaction is specific for the in vitro immunizing erythrocytes. Antibody was demonstrated in the culture fluids.

Stimulation of DNA synthesis in cultures of mouse spleen cell suspensions by bovine transferrin

Abstract Marked enhancement of DNA synthesis was observed in spleen cell suspensions of normal B6D2F 1 mice cultured in the presence of fetal bovine serum. Nearly all of the stimulatory potency of the fetal bovine serum was found to be related to the transferrin fraction. Bovine transferrin isolated from ox serum, which itself did not possess any stimulatory activity, caused enhancement of thymidine incorporation to the same extent as fetal bovine transferrin. No immunological mechanism seems to be involved in the action of bovine transferrin on lymphoid cells, as cells obtained from mice unresponsive to bovine transferrin responded to the same degree as cells derived from normal or sensitized animals.

[29] Use of Sephadex G-10 to separate macrophages and lymphocytes

Publisher Summary This chapter discusses the use of Sephadex G-10 as a column matrix for removing macrophages from suspensions of immunologically reactive cells. The basis of separation involves both adherence and size. Anchorage-dependent cells including macrophages adhere to beads made of Sephadex and large cells may become trapped in the spaces among the relatively inelastic G-10 beads. It reviews that other standard chromatographic grades of Sephadex do not permit efficient recovery of nonadherent lymphocytes because these beads are more elastic and thus pack more tightly, obstructing the flow of even small cells. The chapter explores that the technique has been widely employed to obtain macrophage-free lymphocyte-rich cells for various cellular immunological studies. In contrast to flow cytometry, the G-10 column cell separation procedure is a simple, inexpensive, rapid method for processing relatively large numbers of cells under sterile conditions. Thus, it is particularly suitable as the first step in a more elaborate purification scheme if higher degrees of purity than those produced by G-10 alone are required.

Methods for the study ofin vitro immunization of mouse spleen cells

ConclusionWe have reviewed some of our experiences in developing techniques for studying the functions of the cells of the immune system. It is quite clear that much remains to be done. Improvements in the culture system are needed to permit cells to be grown for longer durations and at lower cell concentrations. The important effects of fetal calf serum should be defined. More sophisticated methods for separating cells into distinct functional populations must be developed. New assays for identifying other functions of the cells, particularly a method for directly assaying the number of precursor cells in a population, are needed. When these techniques are applied to the study of immune cells, further facts should be learned which will permit the development of significant, testable hypotheses on the function and relationships of the cells of the immune system.

USE OF SEPHADEX COLUMNS TO DEPLETE MONONUCLEAR PHAGOTCYES

Publisher Summary Mononuclear phagocytes can be removed from murine and human lymphoid cell suspensions by passage over the columns of Sephadex G-10. This procedure is particularly useful for rapid and gentle processing of large numbers of cells under sterile conditions. The procedure has been employed primarily to evaluate the functions of macrophages in the induction of in vitro immune responses. The preparation of Sephadex G-10 involves placing approximately two liters of saline in a six-liter flask and adding approximately 250 gm of Sephadex G-10. After the Sephadex has settled, one needs to place a beaker under the column and open the stopcock. When all the fluid has penetrated the column, the cells are quickly loaded onto the Sephadex with a Pasteur pipette, taking care not to disturb the top of the column. Once the cells have penetrated the column, small amounts of balanced salt solution–fetal calf serum is continually added until the band of cells is midway down the column.