Cloning by Limiting Dilution of Hybridoma
| Author: Nanci Donacki |
| Source: Contributed by Nanci Donacki |
| Date Added: Tue May 14 2002 |
| Date Modified: Tue Apr 27 2004 |
Materials
DMEM, high glucose (Life Technologies, Inc. #10313-021 or equivalent)
Fetal Bovine Serum (Life Technologies, Inc. #16000-044 or equivalent)
L-glutamine (Life Technologies, Inc. #25030-149 or equivalent)
Hybridoma Cloning Factor (Fisher # IG50-0615)
50 ml sterile centrifuge tubes (Falcon #2070)
15 ml sterile centrifuge tubes (Falcon # 2099)
96 well culture plates (Falcon #3072))
Hemocytometer
Trypan Blue, 0.4% (Life Technologies, Inc. # 630-5150AG)
Multi-channel pipettor and sterile tips
Reagent Reservoir
HT (Life Technologies, Inc. #11067-030)
Procedure
The day before the cloning, refeed 24-well plates or flasks with fresh medium.
Prepare the cloning medium
DMEM
4 mM L-glutamine
20% FBS
10% Hybridoma Cloning Factor
Resuspend the cells to be cloned. Transfer 1 ml to a sterile 15 ml tube. Transfer 50 ml of this suspension to a clean tube for cell and viability counts.
Count the cells and determine the viability. NOTE: The viability must be greater than 80% to continue.
For each hybridoma cell line, calculate the dilutions to give 4 cells/ml, 2 cells/ml and 1 cell/ml in cloning medium.
Label 50 ml tubes for each clone and dilution.
Add medium to each tube according to the calculated dilutions.
Serially dilute each clone to 4, 2, and 1 cell/ml. The final dilution tube should contain 50 ml of cloning medium at 1 cell/ml.
Pour each of the dilutions into a sterile reagent reservoir. Plate 250 ml/well into 96-well plates - 1 plate at 4 cells/ml, 1 plate at 2 cells/ml and 2 plates at 1 cell/ml.
Complete dilutions and plating for each hybridoma cell line.
Place all plates at 37oC, 8-10% CO2. Incubate for 5-7 days.
Microscopically examine all plates to ensure cloning and plating efficiency before refeeding the plates.
