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        Cloning by Limiting Dilution of Hybridoma

        互联网

        915

         

        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.

         

         

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