Borrowed from David Bowtell (bowtell@ariel.ucs.unimelb.edu.au)

Transfer the srongest positive clones to a 24 well plate by scraping the adhering cells with a truncated yellow tip. Bring the volume to 1 ml using the IMDM-m medium. When cells reach confluency, freeze them ( about E6 cells per full well). Scrape them using a blue tip. Add 1 ml of IMDM-m to cells left in the well, let them grow and repeat the freezing. While the cells are growing, save their sup at each freezing. Use the sup to do the other tests required such as western, immunofluorescence and others. (If sup is not used under sterile conditions, add 0.02% azide and keep at 4 C for months.) Transfer the cells to a 25 cm2 flask in 4 ml of IMDM-m and continue to expand as desired. The cells at this point are "addicted" to the macrophage supplement. It is possible to wean them by gradually reducing the amount of MCM. We do it only if very large volumes are needed. After deciding which one are the best clones, it is advisable to reclone them to ascertain their homogeneity (next chapter). The subclones should be checked by all the assays which were used to test the originals. The subclones should be frozen at least twice. If convinced that the subclone is monoclonal, checking the isotype is possible . If not convinced that the hybrydoma is monoclonal, reclone a second time.

Cloning of Hybridoma

Make an accurate cell count of cells in log phase (3 E5 - 1E6 cells / ml). Determine the volume needed which will give you 100 cells. Add the 100 cells to 25 ml of IMDM-m and plate 250 ml of the suspension in each well. Therefore, each well should get 1 cell delivered into it. Clones should be visible and ready to screen in 10 to 14 days.

Ascites Production

7 days before injection of cells, inject 0.5 ml of Pristane (Tetramethyl - pentadecane, Sigma T-7640), intraperitoneally. Prepare cells in log phase. On day of injection, spin 1-2 xE6 cells per mouse. Resuspend in IMDM or PBS at 1-2 xE6 cells/0.5ml (do not leave the cells without serum for more than 1 - 2 hr). Transfer cells to syringe using a 16G needle. Inject 0.5ml per mouse, using a 20G needle, intraperitonealy. Collect ascites when animals are ready (swollen abdomen), 10 days to 15 days after injection. Spin fluid at 3000 g for 10 min. to remove the cells. If there is an oil layer, remove it and discard. Carefully remove the supernatant from the cells. For storage, add sodium azide to 0.02%. Store at -20 C.

Literature

1. Antibodies - A Laboratory Manual; D. Lane, Ed Harlow.

2. Monoclonal Antibodies: Principles and Practice; James W. Goding.

3. Monoclonal antibody technology; Ailsa M. Campbell.