Limiting Dilution - Monoclonal Antibodies



Even though hybridomas are immortal they are unstable unless growing well, particularly rat-mouse hybrids.

Conditions for optimal growth include freshly supplemented medium (10% fetal calf serum and 2mM and cellular contact (feeder cells or hybridoma concentration) / soluble factors.

Thawing Cells

To grow up a clone previously frozen down, first set up a plate(s) of sterile thymocytes at 1 x 10^7 cells/ml and plate at 100µL/well (1 x 10^6 cells/well). These will contain 10^3 –10^4 macrophages, required for their IL-1 and IL-6 production. Preferably leave these overnight to confirm sterility.

The next day, put 8-9 ml of sterile medium into a 10 ml centrifuge tube and place on ice. Thaw out the clone by holding the vial by its lid in a beaker of water taken from a 37°C water bath. When just thawed, transfer the contents (1-2 ml ) into the cold medium (the DMSO used when freezing down the cells can be toxic to cells if they start to turn over).

Resuspension & Plating

  1. Pellet cells at ~400g, 5 min.
  2. Resuspend cells in ~5 ml medium. Count viable cells.
  3. Adjust to 3000 viable cells/ml.
  4. In a 96-well plate add 100µL medium to all wells.
  5. Add 100µL of the counted cell suspension into each well of the first column of the tray

Serially dilute from left to right

This will leave 100µL in each column except the last one.

Add 100µL medium to the rest to make a final volume of 200 µL.


Leave this plate for 7-10 days in a minimal traffic area of the incubator (set at 8% CO2). If the plate gets bumped it may disperse a single clone of cells causing them to appear as more than one clone. At the end of this time, search the plate for single clones. Write these down on a grid of the plate with a little descriptive information about the clone ie: the size.

Testing for Antibodies

Now you need to test the single clones for antibody secretion but the size of the clone to be tested is now important. If the clone is too small, antibody may not be detectable; if the clone is allowed to grow too large the cells at the centre of it may start to die from local nutrient deprivation or suffer from contact inhibition. The cells are only really optimal when they are in exponential growth phase. It is valid for most cells to not allow them to overgrow since in the case of hybridomas, they may suffer "stress" from local nutrient deprivation and become negative/switched off for antibody secretion. The resultant non-secreting cells grow faster than the antibody producing clones and over time, outgrow them.

Maintaining Clones

It is best to feed a small clone and to feed and resuspend medium to large ones. Feeding is done by removing 50µL and replacing with 100µL since there will have been some evaporation over the incubation period. The following day the cells in the resupended wells should have reach exponential growth phase and can be sampled (50µL) for reactivity. Keep the cells in this growth phase throughout and remember to freeze some down for stocks.