Cell Fusion Procedure: SOP #4 by Craig Story Updated 10/18/06, 11/13/06, 1/9/07
Prepare and collect the following needed reagents and supplies day before fusion:
- 500 ml DMEM 20% FBS + supplements (HEPES, Pyruvate, NEAA, 2-ME)
filter to remove particulates from serum which could injure cells. Make sure serum lot works well with new hybridoma growth
- 500 ml above DMEM20 medium plus HAT for selection of fused cells.
- 500 ml DMEM without serum + 2-mercaptoethanol
10 cm dishes, 50 ml falcon tubes, cell strainer Hybridoma enhancer (BioVeris ™ Hybridoma Cloning Factor). Use at 10%. PEG 50% solution (Sigma, Hybrimax) Hyperimmunized mouse boosted 3 days earlier through tail vein injection (or IP 4 days before) 50 mM 2-mercaptoethanol (1000x) Healthy dividing NS-1 cells (or SP2/0 cells) = “Fusion partners”
800g is about 2000 rpm in benchtop swinging bucket rotor. 5 min at 1250 is sufficient. We have used about 2:1 ratio splenocytes to fusion partner Other protocols recommend a 10:1 ratio of splenocytes to fusion partners (108 splenos + 107 NS1) Generally expect about 0.5 - 2 x 108 splenocytes (50-200 million) from one spleen Therefore would need 25-100 million fusion partners (one flask has about 25 million cells, therefore need 2-3 flasks per fusion split 1:2 the day before)
1. Remove spleen aseptically from freshly killed mouse, place in DMEM/serum in falcon tube. Optional, weigh spleen. Remember to save serum sample from mouse. Rinse spleen in DMEM twice before to reduce possibility of microbial contamination. 2. Crush out cells from spleen with syringe plunger, until all large pieces are broken up. Pass splenocytes through 70 µm cell strainer into 50 ml tube, pellet cells 5 min 1250 RPM. 3. Resuspend pellet in 10 ml RBC lysis buffer. Let sit 5 min room temp. Dilute in Serum Free DMEM, count cells while spinning 500 RPM 8 min in large swinging bucket centrifuge. 4. Also collect and count NS-1 cells. Cells can be removed by flushing with 10 ml pipet melted at an angle at the tip. Avoid banging flask. 5. Adjust ratio of splenocytes to fusion partners to get ~2:1 ratio. Place in same tube and pellet as above. 6. Resuspend in serum-free medium. Repeat twice. (Total of three resuspensions in serum free DMEM). Pellet cells. 7. First resuspend pellet gently by vibrating tube, then slowly add 1.0 ml 50% PEG with stirring over 1 minute. PEG can be at room temperature. 8. Incubate in 37 deg bath set up in hood for 90 seconds 9. Add 4.5 ml serum-free medium slowly over the course of 3 minutes, stirring gently. 10. Add 5.0 ml serum-free medium over the course of 2 minutes 11. Add Serum Free Media to bring volume to 50.0 ml 12. Pellet cells 8 min @800 rpm (gentle spin). 13. Remove supernatant, add 2.0 ml HAT supplemented DMEM20 with 10% cloning factor 14. Gradually bring to volume of 50 ml HAT selection medium 15. Plate into 10-12 wells of 6 well dishes, 5 ml per well. 16. Next day, add 3.0 ml of DMEM20 + HAT + 10% HCF to bring volume to 8.0 ml 17. on day 7, carefully remove 3.0 ml from each well and add back 3.0 ml fresh selection medium.
See SOP #8 for Microengraving methods (Screening)
Screen on microwells 10-14 days after fusion. Freeze remaining cells from each well. Clusters of cells should be visible in the 6 well plates. Freeze extra cells not plated on grids at this point as a “backup.” The longer you wait to screen, the greater the chance that fast growing clones will overgrow slower ones.
See SOP #11 for cell picking and expansion/freezing