Mab Production Short Form

Check myeloma cells and prewarm reagents (day of fusion) 5. Use an inverted microscope to check the SP2/0-Ag14 myeloma cells to make sure they are growing vigorously (refractile and not pyknotic), they are not contaminated (no obvious bacteria or fungi), and there are enough cells for the fusion. It is better to postpone the fusion than to perform an ill-advised fusion, since the entire selection and screening effort will take ∼3 weeks.

6. Prewarm the following in a 37◦C water bath: Three 400- and three 600-ml beakers, each containing ∼100 ml H2O 20 ml sterile complete serum-free DMEM 5 ml sterile 50% PEG solution. Harvest spleen and prepare cells 7. Sacrifice boosted animal(s) (UNIT 1.8) and aseptically harvest spleen(s) (UNIT 1.10). Do not use anesthetics for sacrifice. Instead, use cervical dislocation for mouse, or CO2 asphyxiation for mouse, hamster, or rat to avoid introducing an anesthetic into the bloodstream and therefore into the cultures.

8. Transfer spleen to a sterile 100-mm-diameter petri dish filled with 10 ml sterile complete serum-free DMEM. Perform all subsequent steps in a laminar flow hood.

9. Tease spleen into a single-cell suspension by squeezing with angled forceps or by chopping with fine-tipped dissecting scissors. Remove debris and disperse cells further by passage through a fine-mesh metal screen. 10. Transfer spleen cell suspension to a sterile 50-ml conical centrifuge tube and fill with sterile complete serum-free DMEM. Do not use protein- or HEPES-containing medium because the PEG will precipitate proteins and HEPES can be toxic to cells during fusion.

11. Centrifuge 5 min in TH-4 rotor at 1500 rpm (500 × g), room temperature, and discard supernatant. 12. Lyse red blood cells (RBC) by resuspending pellet in 5 ml ammonium chloride solution. Let stand 5 min at room temperature. 13. Add 45 ml sterile complete serum-free DMEM, and centrifuge as in step 11. 14. Resuspend pellet in 50 ml sterile complete serum-free DMEM. Centrifuge as in step 11. Repeat DMEM addition and centrifuging once (each repeat is a wash). 15. While spleen cells are being washed, separately harvest the SP2/0-Ag14 myeloma cells (from step 5) by transferring the cells to 50-ml conical centrifuge tubes. Centrifuge as in step 11. Resuspend myeloma cells in DMEM and pool all cells into one 50-ml conical centrifuge tube.Wash myeloma cells three times as in step 14. 16. Separately resuspend the spleen and myeloma cells in 10 ml complete serumfree DMEM. Count cells and assess viability in each cell suspension using a hemacytometer and trypan blue exclusion (APPENDIX 3B); there should be nearly 100% viability of both suspensions. 17. On basis of cell counts (from step 16), calculate the amount of complete DMEM20/HEPES/pyruvate needed to plate cells at ∼2.5 × 106 total cells/ml. Prewarm this amount of complete DMEM-20/HEPES/pyruvate in 37◦C water bath. Prepare 96-well flat-bottom plates by labeling them sequentially: one plate is required for each 10 ml of final cell suspension. Perform cell fusion 18. Mix SP2/0-Ag14 myeloma and spleen cells at a 1:1 ratio in a 50-ml conical centrifuge tube. Fill the tube with complete serum-free DMEM.

Other cell ratios work. Successful fusions have been performed with a ratio of myeloma/spleen cells as low as 1:20.

19. Centrifuge cell mixture 5 min at 500 × g, room temperature. 20. While cells are in the centrifuge, prepare three 37◦C double-beaker water baths in the laminar flow hood by placing a 400-ml beaker (from step 6) containing 100 ml of 37◦C water into 600-ml beaker containing 75 to 100 ml of 37◦C water. Place the tubes of prewarmed 50% PEG solution and complete serum-free DMEM (from step 6) into two of the 37◦C water baths in the hood. 21. Aspirate and discard supernatant from the mixed-cell pellet (from step 19). 22. Perform the cell fusion at 37◦C by placing the tube containing the mixed-cell pellet in one of the double-beaker water baths in the laminar flow hood. 23. Using a 1-ml pipet, add 1 ml prewarmed 50% PEG to the mixed-cell pellet dropbydrop over 1 min, stirring the cells with the pipet tip after each drop. Stir for an additional minute. 24. Using a clean pipet, add 1 ml prewarmed complete serum-free DMEM to the cell mixture drop-by-drop over 1 min, stirring after each drop. Repeat once with an additional 1 ml of prewarmed complete serum-free DMEM. 25. With a 10-ml pipet, add 7ml prewarmed complete serum-freeDMEMdrop-by-drop over 2 to 3 min. Macroscopic clumps of cells should be obvious at this point.

26. Centrifuge 5 min at 500 × g, room temperature. 27. While the cells are in the centrifuge, rewarm the beaker water baths to 37◦C and place in the hood. Place prewarmed complete DMEM-20/HEPES/pyruvate (from step 17) in the beaker water bath. 28. Discard the supernatant (from step 26). Place tube in the beaker water bath. 29. With a clean 10-ml pipet, forcefully discharge 10 ml prewarmed complete DMEM20/HEPES/pyruvate to the cell pellet. 30. Repeat step 29 until the total volume of prewarmed complete DMEM-20/HEPES (calculated in step 17) is added. If necessary, allow clumps to settle and disrupt with the pipet tip. Further warming of cell suspension is no longer required. If the total volume exceeds 50 ml, gently aspirate and transfer to another sterile container such as a tissue culture flask.

31. Gently aspirate 10ml of cell suspension with a 10-ml pipet. Add 2 drops (100 to 125 µl) of suspension to each well of a 96-well flat-bottom plate (continue until entire suspension is plated). Incubate overnight in a humidified 37◦C, 5% CO2 incubator. Vigorous pipetting of the cell suspension should be avoided at this point, as the newly formed hybrids are unstable. Moreover, the vigorous addition of cells to the wells with repeating micropipettor is not advised. Use a pipet aid and hold the 10-ml pipet at a 45◦ angle with the tip 1 to 2 cm above the well, bracing the pipet with a finger from the opposite hand. To avoid introducing contaminants, do not hold hands above the plate. A steady, even flow of drops from the pipet will allow the most efficient delivery of cell suspension or medium to the wells. Use a fresh pipet to withdraw additional cell suspension. As an optional step to minimize fibroblast overgrowth, permit the fibroblasts in bulkfused cell suspension to adhere overnight to tissue culture flasks before seeding the 96-well plates. Many investigators select their hybridomas under bulk conditions—i.e., they incubate large numbers of cells per well in larger plates or flasks. This makes feeding easier, but allows fast-growing hybridomas to overgrow the others. Since nonproducing hybridomas

tend to grow faster, especially in the hamster-mouse fusions, hybridomas are isolated initially in multiple small wells in this protocol. The primary hybridomas tend to be monoclonal. This is especially important when screening procedures are used that require differential reactivities, e.g., to different cell lines by flow cytometry analysis or to different antigen preparations. In those cases, multiple hybridomas per well will obscure the reactivity of the MAb of interest.