Subcloning Positive Hybridomas

After a positive clone has been found during the screening and has been expanded to 24 well plates or larger volumes, the cell line is ready to be subcloned. This is done for positive-testing cells so that if there is more than one clone in the original well, you can dilute the population to one or a few cells and regrow, to acheive a purely monoclonal culture. Subcloning is done at least twice to ensure a completely monoclonal colony.

Preparation for Subcloning

For every cell line you wish to subclone, you need to prepare a 96-well plate with 50 µL of feeder layers in each well. Let grow overnight (max. 1 week) to check for bacterial contamination. Your expanded hybridomas need to be a large colony of rapidly growing, healthy cells (>90% viability).

 

Subcloning

Dilution of cells

  1. Count the cells on a hemocytometer, then dilute in DMEM/20% FBS so that you have 15 mL of 50 cells/mL (this usually calls for two dilutions, one to get the cells to 50,000 cells/mL, then another 1:999 dilution).
  2. Take 1 mL of the 50 cells/mL and dilute 1:9 with DMEM/20% FBS to get 5 cells/mL.

Plating, Growth, and Screening

  1. With a multichannel pipet, plate 200 µL/well of the two dilutions on top of the feeder layers. Use the first six columns for the 50 cells/mL (giving 10 cells/well) and the last six columns receive the 5 cells/mL (giving 1 cell/well).
  2. Place in incubator and let grow 7-10 days, monitoring the growth. At about day 4, look at the wells and note which ones appear to only have one colony growing in them (i.e. only one clumping of hybridomas). The wells with the 10 cells/well dilution will have many colonies, and grow faster. The wells with 1 cell/well are the ones you are interested in. Screen them when they are around 50% confluent (covering half the bottom of the well) and look for positives. What you hope for is that all wells that have cells in them will test positive--this means your line is monoclonal. If none of the 1 cell/well dilution wells make it, then screen the wells with the 10 cells/well dilution.

Expansion and Freezing

  1. Expand into a 24-well plate a few colonies that test positive in the screen and that you identified early on as having only one colony.
  2. Subclone at least one more time, and as many times as necessary so that all growing colonies test positive in the screen.
  3. Expand into a T25. Then expand into a T75 with only DMEM/10 %FBS and no feeder layers. Freeze these expanded colonies.

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