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An Improved Primary Response from Mouse Spleen Cells Cultured in Vivo in Diffusion Chambers¹

From The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Abstract

The optimum conditions for the hemolytic plaque-forming cell response of mouse spleen cells to sheep red blood cells (SRBC) in Millipore diffusion chambers were determined because of the wide applicability of the technique as an in vivo but closed system for the study of the immune response. The effect of pretreatment of chamber recipients with x-rays or cyclophosphamide, as well as the effects of chamber volume, culture medium, chamber detoxification and sterilization techniques, were investigated. The improvement in response over that previously reported by this laboratory was attributed to larger chamber volume, chamber detoxification twice over a 24-hr period, culture medium supplemented with horse serum, and lethally irradiated or cyclophosphamide-treated recipient C31F₁ mice. With the improved conditions the peak primary response of mouse spleen cells to SRBC (10⁴ direct plaque-forming cells per 10⁶ nucleated cells harvested) was almost 20-fold greater than that obtained in the cell transfer system and about 5-fold greater than the best response obtained both in vitro and in situ. The burst size was also significantly larger than that obtained in the other systems, and culturing efficiency was twofold greater than previously reported from this laboratory for the diffusion chamber technique. The improved technique was used to determine the response of mouse spleen cells to SRBC in both allogeneic (across H-2 and non-H-2 barriers) and xenogeneic hosts. The enhanced response obtained by the present method is attributed to increased culturing efficiency and burst size, but not increased efficiency of interaction of the cells initiating the response.

Footnotes

¹ This investigation was sponsored by the United States Atomic Energy Commission under contract with Union Carbide Corporation.

² Oak Ridge Graduate Fellow under appointment from the Oak Ridge Associated Universities.

³ Present address: Gerontology Research Center, Baltimore, Maryland 21224.

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