Thymocyte-conditioned medium

Homogenize the spleen to produce single cells, and then resuspend them in 10 mL of the thymocyte-conditioned medium. 

Thymocyte-conditioned medium (MTM), 44-45 for in vitro immunization, 9 use in cell fusions, 44 use in stabilizing hybridoma clones, 44... 

This technique is performed in tissue culture and works well when only small quantities of antigen (1-2 pg) are available. It also allows the production of antibodies to substances that are toxic in whole animals. There is no immunological processing of antigens so only soluble, simple antigens such as peptides can be used for this approach. A source of interleukins-4 and -5 is required for the method to work and the easiest way of obtaining them is from thymocyte-conditioned medium. 

Micklem, L. R., McCann, M. C., and James, K. (1978) The use of rat mixed thymocyte-conditioned medium for hybridoma production, cloning and revival. J. Immunol. Methods 104, 81-86. 

Reading (1982) adapted this to obtain concentrates of NSF for in vitro immunization. For this purpose, thymocytes are prepared (Section 5.4.2.4) from both BALB/c and C57BL/6 mice and equal numbers are mixed to a final concentration of 5x 10 cells/ml and cultured in plastic culture flasks in DMEM supplemented with 4.5 g dextrose/1, MEM non-essential amino acids, sodium pyruvate, 2-ME, HAT, HEPES, and 2% rabbit serum. After 48 h, the cells and other debris are pelleted and the supernatant (thymocyte-conditioned medium, TCM) is filtered (200 nm) and stored at — 70°C in aliquots of 10 ml. 

Allogeneic mixed thymocyte medium (4) is a conditioned medium used to aid cells recover from the fusion process. It also encourages division of hybridoma cells after dilution cloning when colonies derived from single cells are required. Rat thymi are used for this purpose to minimise the number of animals required to produce adequate numbers of cells. The medium contains a number of helper cytokines produced by the thymocytes. It is important that the thymocytes used are derived from two different rat strains as this causes co-stimulation and enhanced cytokine secretion by the cells. Mixed thymocyte medium must always be diluted with RPMI 1640 medium containing 15% FBS and is usually used at a dilution of 10-15%. 

Mixed leukocyte (or thymocyte) culture-conditioned medium Thymocytes produce lymphokines which stimulate and support growth of B cells (Andersson et al., 1977). These factors may be non-specific (NSF) or specific for the immunogen (Kilburn and Levy, 1980). These NSF represent a heterogeneous group of molecules... 

M37. Ming, J. E., Cernetti, C., Steinman, R. M., and Granelli Piperno, A. Interleukin-6 is the principal cytolytic T lymphocyte differentiation factor for thymocytes in human leukocyte conditioned medium. J. Mol. Cell. Immunol. 4, 203-211 discuss. (1989). 

Finally, Wenzel et al. (20) examined the role of MCP-1 in an anti-thymocyte antiserum-induced model of GN. Using a neutralizing polyclonal antiserum against rat MCP-1, they demonstrated a significant reduction in macrophage, but not neutrophil, infiltration into the glomeruli 24 h after induction of disease. In addition, the monocyte chemotactic activity present in conditioned medium from inflamed glomeruli was reduced to control values in rats treated with the anti-MCP-1 serum. 

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