1. Transfer hybridoma cells from the 96-well cloning culture plate to a 24-well culture plate in 1 mL of fresh CM (see Note 4) using gentle pipetting to dislodge the cells from the plastic (see Note 5).
2. Two days later, transfer the cells to a 25-cm2 flask in 2 mL of fresh CM (see Note 6). The next day, add 3 mL of CM to feed the cells.
3. The following day, gently pipet the 5 mL of medium in the flask several times several to dislodge the cells and transfer the total volume to a 75-cm2 flask. Add a further 5 mL of fresh CM immediately to the flask to feed the cells. Add 10 mL of fresh CM 24 h later. The subsequent day, transfer the 20 mL containing the cells to a 225-cm2 flask using gentle pipetting as before, then add 30 mL of fresh CM immediately.
4. The next day, dislodge the cells from the flask by pipetting and add 10 mL of the resulting suspension to each of five 225-cm2 flasks. Add 40 mL of fresh CM per flask. This protocol of passaging 10 mL of cell suspension at a 1:5 ratio can be followed for expansion of hybridomas either for generating cells for high density cultures (Subheading 3.2.) or for preparation of cell banks (Subheading 3.3.). Check the cells for Mycoplasma contamination (see Note 7) and analyze the supernatant for antibody production (see Note 8) before progressing to either of the next stages.
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