Fusion and Cloning

1. Medium A - Pre-fusion Medium and Hybridoma Expansion Medium (StemCell Technologies, Inc. - # 03801)
2.   Medium B - Fusion Medium (StemCell Technologies, Inc. - # 03802)
3.   Medium C - Hybridoma Recovery Medium (StemCell Technologies, Inc. - # 03803)
4. Medium D - Hybridoma Selection Medium (StemCell Technologies, Inc. - # 03804)
5.   Medium E - Hybridoma Growth Medium (StemCell Technologies, Inc. - # 03805)
6. PEG Solution (StemCell Technologies, Inc. - # 03806)

Materials

1.   50 ml Sterile conical tubes (Falcon #2070)
2. 15 ml Sterile conical tubes (Falcon #2099)
3. 10 ml sterile pipets (Falcon # 7551)
4.   1 ml sterile pipets (Falcon #7521)
5. Pasteur Pipets, sterile
6. 100 mm sterile Petri Dishes (Falcon #1009)
7. 96-well culture dishes (Falcon # 3072)
8. 24-well culture dishes (Falcon # 3047)
9.   Forceps
10. Scissors
11. Multi-channel pipettor, 50-200 m l
12. Pipet tips, sterile
13. Reagent Reservoir, sterile
14. Tupperware container

Myeloma Cells

1. One week prior to the fusion, split myeloma cells into Medium A to ensure that they are well adapted.
2. Grow up approximately 2 x 107 healthy cells, in mid-log phase, for each fusion.

 Fusion

1. Count the myeloma cells and resuspend to 2 x 107 cells in 30 ml Medium A in a 50 ml tube.
2. Sacrificed the mouse, saturate in ethanol, and remove the spleen.
3. Place the spleen in a Petri dish containing 10 ml of Medium A.
4. Prepare a single cell suspension of the spleen.
5. Using a Pasteur Pipet, transfer the spleen cells to a 50 ml tube.
6. Rinse the Petri dish with another 10 ml of Medium A and add to the tube.
7. Allow the tube to sit for approximately 1 minute to settle the larger pieces of tissue.  Transfer the cell suspension to a clean tube, leaving behind the larger pieces of tissue.
8. Add 10 ml of Medium A to the tube to wash the tissue pieces.  Allow to settle.  Transfer the medium to the clean tube, combining it with the previous cell suspension.
9. Centrifuge the splenocyte suspension at 400 g for 10 minutes, RT.
10. Resuspend the cells in 10 ml of Medium A and count.
11. Combine 108 viable spleen cells with 2 x 107 myeloma cells in a 50 ml tube.  Centrifuge at 400g for 10 minutes.
12. Discard the supernatant and wash the pellet twice with 40 ml Medium B, pre-warmed to 37oC.
13. Discard the supernatant.  Tap the bottom of the tube to loosen the pellet.
14. Add 1 ml of PEG solution to the pellet over a 1 minute period, continually stirring the cells.
15. Continue stirring for an additional 1 minutes.
16. Stop the fusion by adding Medium B while constantly stirring.
    1 ml over 1 minute
    3 ml over 1 minute
    10 ml over 1 minute
17. Incubate for 5 minutes in a water bath at 37oC.
18. Slowly add 40 ml of Medium A.
19. Centrifuge the cells at 400 g for 7 minutes.
20. Discard the supernatant and wash the cell in 40 ml of Medium A.
21. Slowly resuspend the pellet in 10 ml of Medium C.
22. Transfer to a T75 flask containing 40 ml of Medium C.
23. Incubate 16-24 hours at 37oC, 5 CO2.
24. Thaw Medium D and mix.
25. Transfer the cells from the flask into 2x50 ml centrifuge tubes and centrifuge at 400 g for 10 minutes.
26.   Discard the supernatants and tap to loosen the pellets.
27. Combine the pellets and transfer the cells to Medium D.  Mix gently by swirling the tube.
28. Let sit for 30 minutes at 37oC, 5 CO2.
29. Plate 9.5 ml of cells into 10-100 mm Petri dishes.  Tilt the plates to level the mixture.
30. Transfer the plates to a Tupperware container containing a Petri dish with 10 ml sterile water.
31.   Incubate plates at 37oC, 5 CO2.

1. After 10-14 days, examine the plates for the presence of colonies.
2. Remove isolated colonies from the plates using a pipettor with a sterile tips.  Set the pipettor for 10 m l.
3. Pipet each colony into a separate well of 96-well plates contain 200 m l of Medium E.
4.   Incubate the plates at 37oC, 5 CO2 for 1-4 days without feeding.
5.   Remove the supernatants from the wells and test.  Refeed the wells with 200 m l Medium E or other Hybridoma expansion medium.

  Recloning in ClonaCell-HY
Note:  More than 95 of the colonies will be monoclonal when selected by ClonaCell-HY.  Recloning can be done to ensure stability.

1. Once the cells are growing well in 24-well plates, resuspend the cells with a 1 ml pipet.
2. Remove 10 m l of the cell suspension and add to 1.0 ml of Medium A.  Mix well.
3.   Remove 100 m l of this suspension and add to a tube containing 10 ml of Medium D.  Mix well and add to a 100 mm Petri dish.
4. Spread evenly by tilting the plates.  Incubate at 37oC, 5 CO2 as previously described.
5. Repeat for each clone.
6. After 10-14 days, select colonies and transfer to 96-well plates before testing.