EuroMAbNet: European Monoclonal Antibodies Network

Protocols

Cloning method

The objective of cloning the cells producing the antibody of interest is to ensure that the desired hybridoma cell line produced is obtained from a single fused cell.  After a fusion, many different hybrid cells will be present in a single well resulting in the growth of multiple colonies in each well. Your specific antibody-secreting colony is therefore likely to be mixed with other cells that are either non-secreting or which are producing an antibody of undesired specificity.

Figure – Outline showing the various major stages of the cloning methodology.
The purpose of the cloning technique is to grow a clone of cells from an isolated single cell and is outlined above. The method that we use for cloning is that of limiting dilutions. This means you dilute the cells to a concentration calculated so that each well of a 96 well plate will contain one, two or no cells after plating out. Once the cells have begun growing to form a clone, you can view each well under the microscope and discount any well with more than one clone or no clones at all.

Method

• Thaw your cell line a few days before cloning.
•  Ensure that the cells are dividing rapidly before cloning.
• Make up the cloning medium as follows:
  • RPMI 1640 medium with L-glutamine, (Lonza BE12-702F)
  • +10% FBS (previously heat-inactivated) ( Genycell GCS0101-500)
  • + Penicillin (100U/ml) /Streptomycin (100mg/l) ( Gibco 15950-017)
  • + Ultroser G (1%) ( Pall 15950-017)
  • + HAT (Hypoxathine, Aminopterin and Thymidine) supplement which is usually 50X (dilute 10ml in 500mls of medium) (Gibco 21060-017)
  • + 1% or 2% Condimed ( Roche 11088947001)
• Mix the cells in the flask well and perform an accurate count of the cell number.
• Place 10μl of the cells into 10 ml of cloning medium in a 15ml centrifuge tube – this is the master mixture for diluting.
• For one plate of cloning at one cell/ 200 ml medium per well you will need a total of 80 cells in 16 ml cloning medium i.e. a cell concentration of 5 cells/ml.
• For example: A concentration of 10x10⁴ cells in 1 ml in the  culture flask is equivalent to 10 x10² cells in 10μl or 1000 cells in 10μl. Adding this 10 ml to the cloning medium means that you will have a concentration of 100 cells/ml in the master cloning mixture.  Adding 800 μl of this master mixture (i.e. 100 cells) to another 15.2 ml of cloning mixture will result in a dilution of 5 cells/ml and the correct dilution for plating out. 
• Mix the cells well, but very gently just before you pipette them into the microtitre plate.
• Add 200µl to each of the 80 wells in a 96 well microtitre plate.
• Leave in culture at 37ºC for approximately 5-7 days.
• Do not disturb the plate during this time as this may move cells within the wells and give the appearance that there are no single clones.
• View the clones using a microscope. Look carefully at each well for single clones and mark these wells to distinguish them from doubles/triples.
• Leave the clones to grow to about 1/3rd of the size of the well and then test in the appropriate manner (e.g. using an ELISA or immunocytochemical staining technique).
• Test only single clones.
• Expand the positive clones slowly moving them into 2ml wells still using the cloning medium.  Do not mix the contents of the different original microtitre well contents as these constitute potential different cell lines.
• Expand them gradually, (do not rush them), into small flasks.
• It is advisable to retest the supernatant at this point to ensure that the hybridoma cells are still secreting the required antibody.
• Freeze down the hybridoma cell lines.
• If you are not sure if the positive well was a single clone you must reclone the line a second time until you are sure you have achieved a cell line that has been cloned from a single cell.

Using the cloning method to test the quality of your FCS/FBS batch

It is extremely important to use good quality FCS/FBS when trying to produce or grow hybridoma cells.  To test the quality of a potential new batch of FCS/FBS it is recommended that you clone a hybridoma cell line, using the method above, in the FCS/FBS you are currently using and in the new serum test batches.  You would expect to find that the new batch had a cloning efficiency similar to or slightly higher than your original batch.  If you have no previous batch for comparison, you should select a batch that has a cloning efficiency of at least 20% and which results in subsequent healthy cell growth.