Allogeneic Mixed Thymocyte Conditioned Medium

1. Obtain two 6-wk-old rats of different strains (e.g., Sprague and Wistar).

2. Kill them and remove the thymus glands aseptically.

3. Homogenize the thymus glands using the frosted ends of glass microscope slides (see Subheading 3.3. for method of spleen hemogenization) and suspend in 10 mL of phosphate-buffered saline (PBS).

4. Wash by centrifugation at 400g and resuspend in 10 mL of PBS.

5. Add the thymocyte suspension to 1 L of RPMI 1640 medium containing 15% FBS and distribute into 4X 225-cm T flasks.

6. Incubate the cells for 30 to 40 h at 37°C/5% CO2 (it is important that incubation is not longer than 40 h or undesirable cytokines will be produced).

7. Harvest the conditioned medium is by centrifugation at 500g. The medium should be tested for sterility and stored at -20°C in 20-mL aliquots.

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