Protein Protocols Protein Protocols

Hybridoma Production 1113
15. After 10–14 d, feed the wells containing hybridomas with warm HT medium (2 mL 50X
HT + 98 mL 15% FBS/RPMI).
16. Screen the wells containing hybridomas for antibody secretion (see Chapters 160 and 161)
when the medium becomes yellow-orange (acid) and hybridomas are nearly confluent.
17. Expand positive wells into further plates or bottles (after a further 5–8 d, the cells will
grow in medium containing 10% v/v FBS). Clone or cryopreserve in liquid nitrogen.
18. To cryopreserve, resuspend about 10 7 hybridomas in 0.5–1.0 mL of medium containing
20% v/v FBS and 10% v/v DMSO in cryotubes. The freezing mixture should be cooled to
approx 4°C during use by storing the solution in a refrigerator and placing the cryotubes in
ice water. Slowly freeze the cells in nitrogen vapor for at least 2 h, followed by storage in
liquid nitrogen (see Note 3). Hybridomas are thawed rapidly in a 37°C water bath, washed
in ~50 mL medium to remove DMSO, spun down, and resuspended in fresh medium/FBS.
3.2. Cloning (see Note 4)
1. Weigh out 0.5 g agar into a 100-mL autoclave bottle.
2. Add 10 mL distilled water, and autoclave for 15 min.
3. Transfer to a water bath set at 50–55°C, and mix with 90 mL warmed medium (15% FBS/
RPMI at 50°C).
4. Add 12–14 mL of the agar solution to each Petri dish, and allow to set for 20 min at room
temperature (make up three dishes for each line to be cloned).
5. Suspend hybridomas in a well or flask, and transfer 2–4 × 103 cells in 0.2 mL of medium
to one well of a fresh 24-well plate. Add 0.8 mL medium to this and make two more serial
1:5 dilutions (i.e., 0.2 + 0.8 mL medium). Discard 0.2 mL from the final well.
6. Allow the agar solution to cool to about 45–47°C, and add 1 mL of the agar to each well.
Mix and transfer the contents of each well into separate agar containing Petri dishes (avoiding
introducing bubbles). Incubate at 37°C in a CO2 incubator.
7. Check daily for cell growth with an inverted microscope, and discard plates containing
overgrown or no cells. When clones grow into colonies of 20–100 cells, pick them individually
from the plate using a Pasteur pipet, and transfer into 1 mL of medium in separate
wells in a 24-well plate.
8. Feed every 2–4 d, and when cells are 75–100% confluent, test for antibody secretion (see
Chapters 160 and 161) and expand positive wells. Hybridomas may be weaned onto 10%
FBS/RPMI at this stage.
9. Repeat the cloning procedure.
4. Notes
1. Test several batches of FBS before ordering a large quantity. Check for toxicity and sterility,
and if possible, compare with a previous batch of FBS that is known to support hybridoma
growth. Alternatives to FBS are available, such as CLEX, Dextran Products, Canada.
2. To reduce the possibility of contamination of the NS0 line with mycoplasma, bacteria,
fungi, yeast, or virus, always:
a. Use a separate bottle of medium for NS0.
b. Do not manipulate other cell lines in the hood at the same time as NS0.
3. Do not leave cells in DMSO at room temperature since they will die.
4. If hybridomas are in HAT medium, carry out cloning in HT medium.
Reference
1. Köhler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature 256, 495.