细胞培养，鉴定，融合，流式
核酸转染，检测
HYBRIDOMA PRODUCTION
Materials
- Tumor cells that have been treated with 8-azaguanine for 48 hours are removed from the drug and grown to a maximum concentration of 500,000 cells per ml.
- Rats or mice that were previously immunized and then boosted IV 72 hours prior to hybridization.
- Media: Auto-Pow (powdered MEM from Flow Labs) with sodium bicarbonate plus non-essential amino acids, penicillin-streptomycin, L-glutamine and HT. For the serum-containing media (used for the final plating), add 5 - 10% newborn calf serum.
- 40% PEG: Aldrich 1000. Made up in serum-free medium (SFM). The stock may be aliquoted and stored at -30°C.
- Other materials include: 96-well plates, sterile Petri dishes, conical 15 and 50 ml tubes, round bottom (3 and 12 ml) tubes, sterile dissection instruments, 37°C water bath, spleen crusher, 70% ethanol, syringe and needle.
Procedure (Keep all cells and solutions at room temperature or 37°C)
Day 0
· Preparation of Tumor Cells
· Preparation of Spleen Cells
· Cell Fusion
Day 1
Feed the cells by adding an additional 0.1 ml per well with media containing serum and HT plus 2x methotrexate. Process and store the antisera.
Day 3
Replace 0.1 ml of media from each well with 0.1 ml of fresh HT media.
Day 7
Repeat the Day 3 procedure.
Day 11
Repeat the Day 3 procedure, and continue to feed every 7 days.
Screen the wells when the media just begins to turn from the usual orange color to yellow. This insures that the cells have grown to a number adequate to produce detectable antibody but that they not in danger of overgrowing. The screening typically occurs between days 11 - 14. |
