Cloning by Limiting Dilution of Hybridoma

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.  Hybridoma Cloning Factor (Fisher # IG50-0615)
5. 50 ml sterile centrifuge tubes (Falcon #2070)
6. 15 ml sterile centrifuge tubes (Falcon # 2099)
7. 96 well culture plates (Falcon #3072))
8.  Hemocytometer
9. Trypan Blue, 0.4% (Life Technologies, Inc. # 630-5150AG)
10. Multi-channel pipettor and sterile tips
11. Reagent Reservoir
12. HT (Life Technologies, Inc. #11067-030)

  Procedure

1. The day before the cloning, refeed 24-well plates or flasks with fresh medium.
2. Prepare the cloning medium
   DMEM
   4 mM L-glutamine
   20% FBS
   10% Hybridoma Cloning Factor
3. 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.
4. Count the cells and determine the viability.  NOTE:  The viability must be greater than 80% to continue.
5. For each hybridoma cell line, calculate the dilutions to give 4 cells/ml, 2 cells/ml and 1 cell/ml in cloning medium.
6. Label 50 ml tubes for each clone and dilution.
7. Add medium to each tube according to the calculated dilutions.
8. 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.
9. 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.
10. Complete dilutions and plating for each hybridoma cell line.
11. Place all plates at 37^oC, 8-10% CO₂.  Incubate for 5-7 days.
12. Microscopically examine all plates to ensure cloning and plating efficiency before refeeding the plates.