- Anaesthetised rats immunised at D0, D10 and D17 by foot-hock injection. All immunisations with 50ul of cells in RPMI-1640 with D0 injection also performed with Incomplete Freund’s.
- Popliteal LN removed under sterile conditions on D21. NS-1 cells have previously been grown to confluence. NS-1 cells and LN cells are added at a 1:1 ratio and centrifuged @1500rpm, 5min., 4 oC.
- Supernatant is removed and 0.8.mL of PEG4000 in RPMI-1640 is added at a constant rate over 30-45 seconds with vortexing. Note, PEG is made up previously, autoclaved and immediately placed in water bath to prevent solidification.
- Cells are incubated for 3min. @ 37oC followed by addition of RPMI: 2mL over 2min. followed by 8mL over 1min. (syringes placed in incubator prior to addition).
- Cells are centrifuged and resuspended in 40mL of RP10 supplemented with 2X HAT medium and 10% CAS. Cells are then plated out at 100ul/well in 96 well flat-bottom plates containing 10^6 thymocytes/well in 100ul of RP10. These plates are prepared previously to ensure sterility.
- Fusion plates are sealed in glad wrap and placed in a 37oC, 8% CO2-in-air incubator. On D7, 100ul of media/well is removed and replaced with 100ul of 2X HT media. Screening for positive clones is begun around D10 post-fusion.