Generation of Hybridomas
Steve Roffler
Institute of Biomedical Sciences
Academia Sinica
Taipei, Taiwan
Email: sroff@ibms.sinica.edu.tw
Immunization
i. Soluble proteins
Inject 3 week old Balb/c mice with 50-100 mg antigen in 200 ml complete Freund's adjuvant (CFA) in back s.c.
↓ 2-3 wk
repeat immunization in IFA
↓ 7 days
check titer
↓ 1-2 wk
final boost, 50 mg antigen i.v.
↓ 2 days
fuse
Additional boosts in IFA can be given every 3-4 weeks before final boost. Use progressively less antigen (ie 50, 30, 20 mg) to preferentially activate high affinity clones.
ii. Particals (cells, virus, etc)
Inject 3 week old Balb/c mice i.p. with 5x106 cells
↓ 2-3 wk
Boost with 106 cells i.v. + 5x106 i.p.
↓ 7 days
check titer
↓ 1-2 wk
final boost, 106 cells i.v. + 107 i.p.
↓ 2 day
fuse
Fusion
Materials
Lipopolysaccharide from E. coli 026:B6 (Sigma)
Basic Medium
DMEM with glutamine
HEPES 2.98 g/L
NaHCO3 2.00 g/L
Penicillin 100 U/ml
Streptomycin 100 mg/ml
PEG 4000 (warmed to 37oC) Boehringer
HAT (50 X, Sigma) HT (500 X, Boehringer)
Basic medium Basic medium
Hypoxanthine 5 mM Hypoxanthine 50 mM
Aminopterin 0.02 mM Thymidine 8 mM
Thymidine 0.8 mM 15% FCS
15% FCS 5% CM
5% CM
Macrophage conditioned medium (CM)
1. Grow P388D1 cells (ATCC) in DMEM + 5% FCS to confluence
2. Change medium to DMEM + 1% FCS + 1 mg/ml LPS
3. Culture cells 2-3 days (yellow-orange) and collect conditioned medium
4. Filter sterilize, aliquot, and freeze -80oC.
Method
1. Four days before fusion, prepare HAT, HT, and Basic medium.
2. Check myeloma cells (FO cells, ATCC) for sensitivity to HAT (only necessary for each batch of frozen FO cells).
3. Culture myeloma cells in Basic medium with 5% BS (bovine serum). Viability must be > 95% before fusion.
4. One day before fusion, prepare thymocytes.
Remove the thymus from three Balb/c mice (3-4 wks)
Prepare single cell suspension in 50 ml HAT
Culture ON, check for contamination
5. On day of fusion, recover myeloma cells (FO) from plate or flask with versene buffer (no trypsin). Spin down 6x107 cells and resuspend in 10 ml Basic medium (without serum). Place in CO2 incubator while getting spleen.
6. Remove the spleen from the immunized mouse and place in 5 ml basic medium. Prepare single cell suspension in 10 ml Basic medium. There should be about 2x108 cells.
7. Combine FO and spleen cells in 40 ml Basic medium. Wash with 40 ml warm Basic medium (w/o FCS) 3 times. All centrifugations at 1400 rpm for 5 min.
8. Totally remove medium after last centrifugation.
9. Place tube in 37oC water in thermos 1
10. Add 1 ml PEG 4000 (preheated in incubator) over 1 min with gentle mixing.
11. Continue mixing 1 min
12. Add warmed Basic medium w/o FCS as follows:
2 ml over 2 min
2 ml over 1 min
7 ml over 2 min
12 ml all at once
13. Centrifuge at 800 rpm for 5 min. Discard sup.
14. Transfer cells to large culture dish in 110 ml HAT. Add 50 ml of thymocytes (160 ml total).
15. Aliquot 200 ml per well in a total of eight 96-well culture plates.
16. Screen for antibodies after 7-10 days culture in a humid CO2 incubator. Usually 100 ml HAT medium is removed from each well with a eight channel pipette on day 5 or 6 and 120 ml fresh HAT medium is added. Screening can then take place 2-3 days later
17. Transfer hybridomas from positive wells to 24 well plates in HT medium. Culture to near confluence and rescreen to ensure stable secretion of antibody.
18. Reclone positive hybridomas in HT medium. Use one 96-well plate per hybridoma. The day before cloning, 1 thymus (pushed through mesh as for fusion) per four 96-well plates is cultured in 20 ml HT medium. Hybridomas in the 24 well plates are accurately counted and then 60 hybridomas are suspended in 15 ml HT medium plus 5 ml thymocytes and transfer 200 ml/well in the 96-well plates. All hybridomas must be cloned at least 2 times.