Preparation of cells for fusion

Myeloma cells are harvested by centrifugation, washed in scrum-free DMEM. and resuspended at 10 cells/ml. Cells from the spleens or mesenteric nodes of immune animals are harvested by disa egating the lymphoid tissue by passage through a fine stainless steel mesh (tea strainer) using a sterile spatula (Protocol 6). Spleens from immune mice generally yield about 10 cells in total whereas immune spleens and mesenteric nodes fhsm one rat can yield up to 4 X 10 cells (excess cells can be frozen in liquid nitrogen for subsequent fusion). 

The latter often give excellent yields of hybridomas probably because the freezing mixture contains dimethyl sulfoxide which is known to assist fusion of cell membranes. 

Preparation of immune lymphocytes and myeloma cells for fusion 

Centrifuge exponentially growing mouse or rat myeloma cells in 50 ml aliquots for 3 min at 400 g, wash twice by resuspension in serum-free DMEM, count in a haemocytometer, and resuspend in this medium to 1-2 x lO cells/ml. 

Kill immune animals by cervical dislocation or CO2 inhalation, test bleed, and open the abdominal cavity. Remove spleens or mesenteric lymph nodes by blunt dissection. 

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Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

The electrolysis Of fused alkali salts.—Many attempts have been made to prepare sodium directly by the electrolysis of the fused chloride, since that salt is by far the most abundant and the cheapest source of the metal. The high fusion temp. the strongly corrosive action of the molten chloride and the difficulty of separating the anodic and cathodic products, are the main difficulties which have been encountered in the production of sodium by the electrolysis of fused sodium chloride. Attention has been previously directed to C. E. Acker s process for the preparation of sodium, or rather a sodium-lead alloy, by the electrolysis of fused sodium chloride whereby sodium is produced at one electrode, and chlorine at the other but the process does not appear to have been commercially successful. In E. A. Ashcroft s abandoned process the fused chloride is electrolyzed in a double cell with a carbon anode, and a molten lead cathode. The molten lead-sodium alloy was transported to a second chamber, where it was made the anode in a bath of molten sodium hydroxide whereby sodium was deposited at the cathode. A. Matthiessen 12 electrolyzed a mixture of sodium chloride with half its weight of calcium chloride the addition of the chloride of the alkaline earth, said L. Grabau, hinders the formation of a subchloride. J. Stoerck recommended the addition of... 

Mannosylated PAMAM dendrimers have also been used in inhibition studies on HIV infection.85 In this case, dimannosylated generation three and four of the dendrimer (Fig. 18) were prepared and tested for their binding to cianovirin-N (CV-N), an HIV-inactivating protein that blocks virus-to-cell fusion through a high mannose-mediated interaction. These dendrimers were found to be effective for the recruitment of CV-N. 

The problem is that if an individual antibody-producing cell is isolated and grown in culture, its descendants have a limited lifespan that severely limits their use for the routine preparation of monoclonal antibodies. In 1975, Milstein and Kohler discovered how monoclonal antibodies of almost any desired antigen specificity can be produced indefinitely and in large quantities. Their method was to fuse a B lymphocyte producing antibody of the desired specificity with a cell derived from a cancerous lymphocyte tumor, called a myeloma cell, which is immortal. The cell fusion is called a hybridoma, which is both immortal and secretes the same specific antibody originally encoded by the B lymphocyte. 

To demonstrate that preservation of cell viability is due to fusion of IL with the cell membranes, IL were prepared with intraliposomal silver grains. The rationale was that the only way silver grains can enter the treated hypoxic cardiocytes is by fusion of the IL with the cell membrane that resulted in preservation of cardiocyte viability. If endocytosis of the IL is the process of internalization without fusion, then plain liposomes should also show internalization of the silver grains. To prepare such liposomes,silver nitrate was added in a buffer solution during liposome preparation by soni-cation. Liposomes, purified from the non-entrapped silver nitrate by dialysis were dialyzed in 0.12 M NaCl solution and exposed to light for 1 h that led to the formation of fine electron-dense precipitate of silver oxide inside liposomes. 

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