Fusion Protocol

1. Kill immunized mouse by CO₂ asphyxia and wet throughly with ethanol. Aseptically remove spleen and place on sterile cell sieve (screen).
2. Cut the spleen into small pieces with sterile scissors and press through the screen with the end of the plunger of a 1ml sterile syringe into a sterile petri dish containing 10ml of DMEM-10%FCS. This is done gently as pressing too forcefully increases the amount of connective tissue present, increasing the chance that the hybridomas will be overgrown by fibroblasts.
3. Aspriate the cells once up and down a 10ml syringe with a 22g needle.
4. Transfer the cells to a sterile 50ml Falcon tube and allow to settle for 5 minutes.
5. Resuspend NS-1 cells from the tissue culture flasks and decant into 50ml sterile Falcon tubes.
6. Remove the supernatant from the spleen cell suspension, leaving the large clumps and put into a fresh Falcon tube.
7. Pellet the spleen cells and NS-1 cells for 5 minutes at 1200rpm.
8. Remove the supernatants and resuspend the cells separately in 10ml of DMEM-10%FCS.
9. Dilute each cell suspension 1:10 in 1% trypan blue in PBS. Count using the haemocytometer.
10. Pellet the cells for 5 minutes at 1200rpm and combine the spleen and NS-1 cells at a ratio of 10:1 (ie., 10⁸ spleen cells to 10⁷ NS-1 cells).
11. Mix gently and and spin at 1200rpm for 7 minutes.
12. Remove supernatant and add 0.5ml 41.6% PEG₁₅₅₀ to the cells with gentle stirrring over exactly 1 minute. Rock the tube for 2-3 minutes, add 0.5ml 25% PEG₁₅₅₀ over exactly 1 minute and rock the tube for a further 2-3 minutes.
13. Add 4ml of DMEM slowly and add a further 20ml of DMEM-20%FCS gently and incubate at 37^oC for 2-3 hours.
14. Prepare thymus feeder cells. Aseptically remove the thymus from a young mouse, prepare a cell suspension as for spleen cells and place in 50ml of DMEM-20%FCS. The thymus of old or large mice should be prepared in 20ml DMEM-20%FCS.
15. Mix the spleen-NS-1 cells with the thymocytes and transfer 1.0ml aliquots to 24 well NUNC tissue culture trays, or 0.1ml aliqouts to 96 well NUNC tissue culture trays.
16. Check after 24 hours for contamination and discard contaminated plates.
17. Prepare selective HAT medium (ie., DMEM-20%FCS with HAT). Remove most of the medium in the wells without disturbing the cells and replace with 1.0ml of HAT medium.
18. After 4 days replace the medium in the wells with fresh HAT medium without disturbing the cells.
19. Wait and screen visually for hybridomas.

Hybridomas can be tested by ELISA when they cover approximately one third of the well. Do not allow the cells to overgrow as antibody secreting cells will die off first.