After cell fusion, when we have positive wells, the next step is cloning cells. Sometimes, the positive wells have several colonies in the wells. Generally, we consider one of the colonies produces antibodies against the antigen.
We expand the culture from a 96-well plate into a 24-well plate. Add 100 ul of HT medium in a well which showed positive results in the screening assay, and suspend cells. Add 0.5 – 1 ml HT medium in a well of a 24-well plate. Transfer all cell suspension in a 24-well plate. Before we clone cells, we have to check antibody production using an indirect ELISA one more time.
With the positive wells in the second screening, we suspend cells, take 10 ul of the cell suspension into 90 ul of trypan blue solution in a sample tube. The trypan blue stains dead cells to allow counting live cells only. 50 ul of the diluted cells can be loaded in a Hemocytometer. Calculate average cell counts/ml, then multiply by 10 from the cell dilution. Dilute cells with HT medium at 1000 cells/ml. Take 200 ul the cells suspension in 20 ml of HT medium containing 5% Briclone(1). Then, add 200 ul (2 cells/well) of the cell suspension (10 cells/ml) in each well (2, 3).
Incubate at 37C at 5% CO2 gas. After 10 – 14 days, check antibody production by a indirect ELISA using single colony wells.
2. S. A. Fuller, M. Takahashi, J. G. Hurrell, Cloning of hybridoma cell lines by limiting dilution. Curr. Protoc. Mol. Biol. Chapter 11, Unit11.8 (2001).
3. E. A. Greenfield, Single-Cell Cloning of Hybridoma Cells by Limiting Dilution. Cold Spring Harb. Protoc. 2019 (2019), doi:10.1101/pdb.prot103192.