Materials

1.  DMEM, high glucose (Life Technologies, Inc. #10313-021 or equivalent)
2. Fetal Bovine Serum (Life Technologies, Inc. #16000-044 or equivalent)
3.  L-glutamine (Life Technologies, Inc. #25030-149 or equivalent)
4. HAT (Life Technologies, #31062-0211 or equivalent)
5. Hybridoma Cloning Factor (Fisher # IG50-0615)
6. PEG1500 (Boehringer Mannheim # 0783-641 or equivalent)
7. Scissors
8. Forceps
9. 50 ml sterile centrifuge tubes (Falcon #2070)
10. 96 well culture plates (Falcon #3072))
11. 6-well culture plates (Falcon #3046)
12. Hemocytometer
13. Trypan Blue, 0.4% (Life Technologies, Inc. # 630-5150AG)
14. Multi-channel pipettor and sterile tips
15. Reagent Reservoir
16. HT (Life Technologies, Inc. #11067-030)
17. Petri dishes (Falcon #1009)
18. Ethanol, 70%
19. PBS, Sterile (Life Technologies, Inc. # 20012-027)

 Procedure

1. Preparation of Spleen cells
1. Sacrifice the animal and swab the abdominal area in alcohol.
2. Open the abdominal area and locate the spleen.
3. Using sterile forceps and scissors, remove the spleen and place in a tube containing 50 ml sterile PBS.
4. Transfer a spleen to a Petri dish containing 50 ml sterile PBS.  Remove any excess tissue and fat.
5. Wash the spleen by transferring to a 6-well plate containing 5 ml/well sterile PBS.
6. Transfer the spleen to a Petri dish containing 50 ml sterile PBS.
7. Prepare a single cell suspension by teasing the tissue with sterile forceps.
8. Collect the cells into a 50 ml tube.  Wash the Petri dish with an additional 10 ml sterile PBS and add to the tube.
9. Allow the cells to settle for 1 minutes.
10. Carefully remove the cell suspension and transfer to a clean tube, being careful not to disturb the larger pieces of tissue at the bottom of the tube.
11. Wash the tube with 10 ml sterile PBS and allow to settle for 1 minute before transferring and combining with the remaining cell suspension in the tube.
12. Centrifuge at 1000 rpm, 5 minutes, room temperature.
13. Carefully siphon off the supernatant and discard.  Tap the pellet to resuspend.  Wash the cells with 50 ml sterile PBS.
14. Carefully siphon off the supernatant and discard.  Resuspend the cells in 10 ml sterile PBS.
15. Take an aliquot for a cell and viability count.
2.  Preparation of Myeloma Cells.
1. Collect myeloma cells from 2-4 T-150 flasks in log phase of growth.
2. Wash twice with sterile PBS.
3. Resuspend the cells in 10 ml sterile PBS.
4. Take an aliquot for a cell and viability count.
3. Fusion Procedure.
1. Mix the spleen and myeloma cells together in a 50 ml tube at a ratio of 2:1-5:1 (Spleen:myeloma).  Top off with PBS.
2. Centrifuge at 800 rpm for 5 minutes, room temperature.
3. Aspirate all of the supernatant.  Tap the bottom of the tube to loosen the pellet.
4. Slowly add the PEG, dropwise over a minute, using the pipet to stir the cells.
5. Continue mixing for another 60 seconds.
6. Dilute the PEG with sterile PBS:
   1 ml in 1 minute
   5 ml in 1 minute
   10 ml in 1 minute.
7. Tops off the tube with sterile PBS.
8. Centrifuge at 800 rpm, 5 minutes, RT.
9. Discard the supernatant.  Resuspend the pellet in HAT medium to 5 x 10⁵ cells/ml.
10. Plate 200 ml/well to 96 well plates.  Incubate at 37^oC, 8-10% CO₂ for 5-7 days.