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)
- 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.
- 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.
- 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.
- 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.
- Adjust ratio of splenocytes to fusion partners to get ~2:1 ratio. Place in same tube and pellet as above.
- Resuspend in serum-free medium. Repeat twice. (Total of three resuspensions in serum free DMEM). Pellet cells.
- 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.
- Incubate in 37 deg bath set up in hood for 90 seconds
- Add 4.5 ml serum-free medium slowly over the course of 3 minutes, stirring gently.
- Add 5.0 ml serum-free medium over the course of 2 minutes
- Add Serum Free Media to bring volume to 50.0 ml
- Pellet cells 8 min @800 rpm (gentle spin).
- Remove supernatant, add 2.0 ml HAT supplemented DMEM20 with 10% cloning factor
- Gradually bring to volume of 50 ml HAT selection medium
- Plate into 10-12 wells of 6 well dishes, 5 ml per well.
- Next day, add 3.0 ml of DMEM20 + HAT + 10% HCF to bring volume to 8.0 ml
- 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