Materials:  P3X63Ag8.653 murine myeloma or YB2/0 (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 µg streptomycin and 50 µM 2-ME in the absence or presence of HAT or HT for selection (I-20).

Procedure:

        NOTE: All washes are at 1,000 rpm for 10 minutes at 4° C using serum free media

Tissue Collection: 1. Sacrifice animal by CO2 inhalation. 2. Remove peritoneal cells from naive mouse for use as feeder cells by peritoneal lavage. Place cells on ICE. 3. Sacrifice immune animal and remove spleen. Place on ICE.

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. In addition, wash peritoneal cells in I-0 twice, resuspend in I-20 and count. 4. Add an appropriate number of myeloma cells to the entire volume of spleen cells according to the following ratios and centrifuge together.

Fusion Protocol: 1. 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 30 seconds, add 1 ml of 37° C PEG while tapping the side fo the tube to achieve thorough mixing. Over the next 90 seconds continue to mix. After approximately 1 minute 40 seconds stop mixing and fill a 5 ml pipet with warm I-0. When exactly 2 minutes have elasped, dilute the PEG/cell mixture slowly by adding dropwise 1 ml of I-0 over a 1 minute time span. During the next 1 minute, add 2 ml I-0 dropwise. The remaining 2 ml I-0 in the pipet is added during the next 40 seconds. Next use a 10 ml pipet and add 14 ml 37° C I-0 during the last 1 minute period. Bring the total volume to 50 ml using I-20. Centrifuge at 4°C and resuspend in HAT medium at the appropriate volume to bring the cells to the following concentrations:

Alternatively, the cells can be resuspended in I-20 + 2-ME with 150 µl added per well. An additional 50 µl of a 4X HAT solution can be added at 16-24 hours after fusion.

2. Add peritoneal cells to the fused cells at 2.5 x 104 cells per ml (this will result in a plating of 5 x 103 PEC per well).

3. Dispense cells into 96 well plates as follows:                  Cells in I-20 + 2-ME          150 µl per well + 50 µl per well 4X HAT after 18-24hrs

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 µl 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

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

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

Sheehan, K.C.F., J. Calderon and R.D. Schreiber. 1988. Generation and characterization of monoclonal antibodies specific for the human IFN gamma  receptor. J. Immunol. 140: 4231.

Sheehan, K.C.F., N.H. Ruddle and R.D. Schreiber. 1989. Generation and characterization of hamster monoclonal antibodies that neutralize murine Tumor Necrosis Factors. J. Immunol. 142: 3884.