Preparation of spleen and feeder cells

Preparation of spleen and feeder cells The mouse killed 

The necessity for feeder cells for the first few days after fusion is undisputed, but not the nature of these cells. Kohler (1978) used 

Thymocytes are obtained from the thymus of BALB/c mice. Contamination by cutting of the oesophagus or the trachea should be avoided. The glands are rinsed with non-supplemented medium and a cell suspension is prepared by forcing the cells into 10 ml supplemented medium (see below). Cell clumps are dispersed by pipetting and the suspension is diluted to 8 x 10 cells/ml. 

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 

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. 

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