CloningbyLimitingDilutionofHybridoma

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.