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Materials

P3X63Ag8.653 murine myeloma (maintained at < 1 x 106/ml)
50% w/v PEG 1500, warmed to 37° C

Medium

IMDM supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 50 U penicillin, 50 ?g streptomycin and 50 ?M 2-ME in the absence or presence of HAT or HT for selection.

Cell Preparation

1. Tease spleen in ice cold serum-free medium (I-0). Pass cell suspension through a Falcon 70 micron cell filter and suspend in 50 ml of ice cold I-0. Centrifuge and wash cells three times at 4° C in I-0. Resuspend cells after the third wash in 10 ml I-0 and count viable cells. Keep cells on ice.
   
   
2. Concurrently with the spleenocytes, centrifuge and wash myeloma cells three times, using I-0 and resuspend in I-0. Count viable cells.
   
   
3. Add an appropriate number of myeloma cells to the entire volume of spleen cells according to the following ratios and centrifuge together.

Fusion Protocol

Aspirate all supernatant and suspend pellet by tapping the end of the tube. Place tube in container of warm water (37°C). Gradually, over a period of 60 seconds, add 1 ml of 37° C 50% w/v PEG while tapping the side of the tube to achieve thorough mixing. Over the next 60 seconds continue to mix. Dilute the PEG/cell mixture slowly by adding dropwise 2 ml of I-0 over a 2 minute time span. Next use a 10 ml pipet and add 8 ml 37° C IMDM containing 10% FCS over a 4 minute period. Bring the total volume to 50 ml using I-10. Centrifuge at 4°C and resuspend in medium at the appropriate volume to bring the cells to the following concentrations:

Type	Cell Concentration
mouse or rat	1.5 x 106 cells per ml
hamster	0.5 - 1.0 x 106 cells per ml

Add150 μl added per well. An additional 50 μl of a 4X HAT solution can be added at 16-24 hours after fusion.

Feeding Schedule

The day of the fusion is considered day 0. Fusion plates are examined at 24-48 hours for any abnormalities (i.e. bacterial contamination). On day 7, wells are inspected visually and then fed. One half of the volume in each well is aspirated using a sterile pasteur pipet. A new pipet is used for each plate. Wells are fed with 125 ?l of I-20 + 2ME + HAT on days 7, 11 and thereafter as needed, i.e. Mon., Wed., Fri.

Cultures are examined visually at each feeding. Once a majority of wells appear 50% confluent for growth, supernatants are harvested for screening by the investigator. Plates are fed at this time.

**Investigators should provide information on the serum titer of the immunized animal. If the titer is greater than 1:10,000, it may be important to feed the cultures at least 3-4 times prior to the screening. This will essentially "wash away" any antibody release from dying B cells and decrease background Ig levels.

References

Sheehan KC, Ruddle NH, Schreiber RD. Generation and characterization of hamster monoclonal antibodies that neutralize murine Tumor Necrosis Factors. J Immunol 142:3884-3893, 1989.

Sheehan KC, Calderon J, Schreiber RD. Generation and characterization of monoclonal antibodies specific for the human IFN gamma  receptor. J Immunol 140:4231-4237, 1988.

Sanchez-Madrid F, Szklut P, Springer TA. Stable hamster-mouse hybridomas producing IgG and IgM hamster monoclonal antibodies of defined specificity. J Immunol 130:309-312, 1983.

Kohler G, Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975