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.

Species	Spleenocytes: Myeloma
mouse-mouse	5:1
hamster-mouse	4:1
rat-mouse	5:1
T cell fusion	1:1

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:

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

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.

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.