Research > Research Cores > Hybridoma Center
Monoclonal Antibody Development CoreGENERAL FUSION PROTOCOL
P3X63Ag8.653 murine myeloma (maintained at < 1 x 106/ml)
50% w/v PEG 1500, warmed to 37° C
DMEM (4g/L glucose) 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 and Hybridoma Cloning Factor (1% final).
- 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.
- Concurrently with the spleenocytes, centrifuge and wash myeloma cells three times, using I-0 and resuspend in I-0. Count viable cells.
- Add an appropriate number of myeloma cells to the entire volume of spleen cells according to the following ratios and centrifuge together.
|mouse - mouse||5:1|
|hamster - mouse||4:1|
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 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:
|mouse||1.0 x 106 cells per ml|
|hamster||0.6 x 106 cells per ml|
Add 150 μl added per well. An additional 50 μl of a 4X HAT solution can be added at 16-24 hours after fusion.
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. 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.
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