In the second method use longer processing of cages PEG solution. 30-35% of PEG are added to a deposit of cages at the room temperature. Cages centrifuge 2 min., then them leave at the room temperature for 5-7 min. Then they are parted in large volume of the environment with serum, washed and cultivated.
Cloning in semi-fluid an agar agar. It is possible to apply a semi-fluid agar to cloning of hybridomas. Usually the system consisting of two layers undertakes. The lower (firm) layer contains 0,5% an agar agar in the environment of cultivation. To it allow to harden. Then add the second layer – the soft, containing 0,3% agar agar which joins the cloned cages. When using cages of the feeding layer they are sowed in Petri's cup before filling an agar agar, and the environment of cultivation is deleted just before addition of the lower layer of an agar.
There are two main options of addition of PEG, now in use. The first a method consists in the following. Within 1 min. add 1 ml of 50% of PEG solution at 370 C with continuous hashing. Then solution is diluted gradually to 10 ml within several minutes Wednesday without serum then cages centrifuge and resuspendirut in the environment of cultivation.
At = 1 f (= 0,3 to receive reasonable probability only of one clone in a hole, in more than 37% of holes it should not be observed growth of cages. As efficiency of cloning seldom happens equal 100%, cages need to be sowed at the density of 10, 3 and 0,5 cages on a hole. Those tablets on which growth in half of holes is observed, contain the isolated clones.
One of problems of cellular engineering consists in creation of cellular systems with new properties on the basis of cellular interactions. Now immunization methods out of an organism are improved and mechanisms of activation of V-lymphocytes are studied. It is especially important for receiving monoclonal antibodies of the person.
In addition in the certain room it is desirable to have a deep freeze on-700 C and a vessel of the Dewar with liquid nitrogen for storage of cages. For receiving hybridomas it is necessary to acquire also special plastic ware for culture of cages: 96-lunochny tablets with a flat bottom, 24-lunochny tablets, bottles with an area of height of 25, 75 cm2, etc., plastic ware for carrying out immunofermental and radio immunological analyses, environments for cultivation, necessary reactants, the fruit serum of a cow (FSC).
Choice of an experimental animal. Usually for immunization use mice and rats. It is connected with that suitable miyelomny cages of mice and rats are widespread and, besides, does not represent difficulties cultivation of the received hybridomas in an organism of these animals.