Protein Protocols Protein Protocols

1134 Thi Man and Morris
300g. Resuspend the pellet in 5 mL of DMEM/25 mM HEPES, and keep at room temperature.
Usually about 1 × 10 8 cells/spleen are obtained (see Note 7).
9. Add 2.5 mL spleen cell suspension to each 10 mL of myeloma cell suspension, and mix
gently. Centrifuge at 300g for 7 min at 20°C. Remove supernatants with a pipet, and
resuspend pellets in 10 mL DMEM/25 mM HEPES using a pipet. Centrifuge at 300g for
7 min. Remove the supernatant completely from one pellet (use a Pasteur pipet for the last
traces), and then loosen the pellet by tapping the universal gently (see Note 8).
10. Remove the DMEM/25 mM HEPES and the 50% PEG from the water bath just before use.
Take 1 mL of PEG in a pipet, and add dropwise to the cell pellet over a period of 1 min,
mixing between each drop by shaking gently in the hand. Continue to shake gently for
another minute. Add 10 mL DMEM/25 mM HEPES dropwise with gentle mixing, 1 mL
during the first minute, 2 mL during the second, and 3.5 mL during the third and fourth
minutes. Centrifuge at 300g for 7 min. (This procedure can be repeated with the second
cell pellet, while the first is spinning.)
11. Remove supernatant, and resuspend pellet gently in 5 mL of DMEM/20% HS. Place both
5-mL cell suspensions in their universals in the CO 2 incubator for 1–3 h.
12. During this time, take the ca. 80 mL of myeloma-conditioned medium and add 4.5 mL of
HECS, 90 µL of aminopterin, and 180 µL of hypoxanthine/thymidine solutions. Filter
2 × 40 mL through 22-µm filters (47 mm; Millipore [Bedford, MA]) to resterilize, and
remove any remaining myeloma cells.
13. To each 40 mL, add the 5-mL fusion mixture from the CO 2 incubator and distribute in
96-well microtiter plates (4 plates/fusion; 100 µL/well) using a Transtar 96 or a plugged
Pasteur pipet (3 drops/well) (see Note 9). Put the plates from each fusion in a separate
lunch box in the CO 2 incubator and leave for 3 d.
14. On d 4, add to each well 80 µL of DMEM/20% HS supplemented with HECS and 5X HT.
Replace in the CO 2 incubator as quickly as possible.
15. By d 10–14, a high proportion of the wells should have a clear, white, central colony of
cells, easily visible with the naked eye. If you want to select for high-affinity MAb (and
you would be well advised to do so, unless you have some very special objectives), do not
delay screening. If you are using a sensitive screening method, such as ELISA, immunofluorescence,
or Western blotting, you will detect high-affinity antibodies from even the
very small colonies. Do not wait for the medium to turn yellow, or your cells may start to
die (if you are using a less sensitive screen, such as an inhibition assay, you may need to
wait longer). A high proportion of the 768 wells (50–100%) should have colony growth
by 14 d. If it is