Fusion and Cloning
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(StemCell Technologies, Inc. # 03800)
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Medium A - Pre-fusion Medium and Hybridoma Expansion Medium (StemCell Technologies, Inc. - # 03801)
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Medium B - Fusion Medium (StemCell Technologies, Inc. - # 03802)
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Medium C - Hybridoma Recovery Medium (StemCell Technologies, Inc. - # 03803)
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Medium D - Hybridoma Selection Medium (StemCell Technologies, Inc. - # 03804)
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Medium E - Hybridoma Growth Medium (StemCell Technologies, Inc. - # 03805)
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PEG Solution (StemCell Technologies, Inc. - # 03806)
Materials
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50 ml Sterile conical tubes (Falcon #2070)
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15 ml Sterile conical tubes (Falcon #2099)
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10 ml sterile pipets (Falcon # 7551)
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1 ml sterile pipets (Falcon #7521)
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Pasteur Pipets, sterile
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100 mm sterile Petri Dishes (Falcon #1009)
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96-well culture dishes (Falcon # 3072)
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24-well culture dishes (Falcon # 3047)
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Forceps
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Scissors
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Multi-channel pipettor, 50-200 m l
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Pipet tips, sterile
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Reagent Reservoir, sterile
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Tupperware container
Myeloma Cells
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One week prior to the fusion, split myeloma cells into Medium A to ensure that they are well adapted.
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Grow up approximately 2 x 107 healthy cells, in mid-log phase, for each fusion.
Fusion
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Count the myeloma cells and resuspend to 2 x 107 cells in 30 ml Medium A in a 50 ml tube.
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Sacrificed the mouse, saturate in ethanol, and remove the spleen.
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Place the spleen in a Petri dish containing 10 ml of Medium A.
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Prepare a single cell suspension of the spleen.
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Using a Pasteur Pipet, transfer the spleen cells to a 50 ml tube.
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Rinse the Petri dish with another 10 ml of Medium A and add to the tube.
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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.
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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.
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Centrifuge the splenocyte suspension at 400 g for 10 minutes, RT.
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Resuspend the cells in 10 ml of Medium A and count.
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Combine 108 viable spleen cells with 2 x 107 myeloma cells in a 50 ml tube. Centrifuge at 400g for 10 minutes.
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Discard the supernatant and wash the pellet twice with 40 ml Medium B, pre-warmed to 37oC.
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Discard the supernatant. Tap the bottom of the tube to loosen the pellet.
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Add 1 ml of PEG solution to the pellet over a 1 minute period, continually stirring the cells.
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Continue stirring for an additional 1 minutes.
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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 -
Incubate for 5 minutes in a water bath at 37oC.
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Slowly add 40 ml of Medium A.
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Centrifuge the cells at 400 g for 7 minutes.
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Discard the supernatant and wash the cell in 40 ml of Medium A.
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Slowly resuspend the pellet in 10 ml of Medium C.
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Transfer to a T75 flask containing 40 ml of Medium C.
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Incubate 16-24 hours at 37oC, 5 CO2.
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Thaw Medium D and mix.
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Transfer the cells from the flask into 2x50 ml centrifuge tubes and centrifuge at 400 g for 10 minutes.
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Discard the supernatants and tap to loosen the pellets.
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Combine the pellets and transfer the cells to Medium D. Mix gently by swirling the tube.
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Let sit for 30 minutes at 37oC, 5 CO2.
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Plate 9.5 ml of cells into 10-100 mm Petri dishes. Tilt the plates to level the mixture.
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Transfer the plates to a Tupperware container containing a Petri dish with 10 ml sterile water.
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Incubate plates at 37oC, 5 CO2.
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After 10-14 days, examine the plates for the presence of colonies.
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Remove isolated colonies from the plates using a pipettor with a sterile tips. Set the pipettor for 10 m l.
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Pipet each colony into a separate well of 96-well plates contain 200 m l of Medium E.
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Incubate the plates at 37oC, 5 CO2 for 1-4 days without feeding.
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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.
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Once the cells are growing well in 24-well plates, resuspend the cells with a 1 ml pipet.
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Remove 10 m l of the cell suspension and add to 1.0 ml of Medium A. Mix well.
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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.
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Spread evenly by tilting the plates. Incubate at 37oC, 5 CO2 as previously described.
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Repeat for each clone.
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After 10-14 days, select colonies and transfer to 96-well plates before testing.