Selection and cloning of hybridomas on viscous medium

Fusion is performed as in Section 5.4.3.1, and the cell pellet is taken up in 15 ml of Iscove s medium containing 53.3% FBS, 2.66% of HAT stock solution, 1.2 x 10 thymocytes and 2 mg EPS. To this, 25 ml of 0.2% (w/v) MC (Section 5.4.2.6) are added. The tube is then tipped several times and mixed for several seconds. Of this suspension 1 ml aliquots are distributed (with a syringe with an 18-gauge needle) into Petri dishes. Two of these dishes are placed in a 10 cm Petri dish, together with a third dish containing 1 ml distilled water and then placed in a 5% CO2 incubator at 37°C. 

From day 9 after fusion and every other day afterwards up to 4 weeks, the dishes are inspected for clearly visible colonies (not simple clumps of cell debris). A colony can be marked on the bottom of the dish with a fine felt-tip pen beneath the colony. Isolated colonies should be at least 0.5 mm in diameter. They are removed 

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