Methods and results
Establish cells in semi solid culture (Using stable hybridoma line)
Purpose: To adapt cells to a semi-solid media and establish plating conditions for ClonePix FL screening. It is advisable to first establish conditions in a stable hybridoma line as this requires less optimization time than starting with a fresh fusion. Please anticipate a couple rounds of plating for this phase.
* Please refer to the “Starter Guide to Hybridoma” for specific protocols on plating and mixing.
- If frozen cells are to be used, thaw and passage for about 48 hours until viability reaches as least 85%, preferably above 90%.
- On day of plating, spin to concentrate cells to 2 mL. Obtain a count of viable cells.
- Practice plating cells at varying densities into media. It is critical to optimize the seeding densities to ensure successful colony selection and picking. Plate out 3 different densities: 50 cells/mL, 100 cells/mL, 150 cells/ mL.
- CloneMatrix+ liquid hybridoma media concentrate (2X). CloneMatrix is the semisolid concentrate. The liquid media should be a media known to support your hybridoma line.
- CloneMedia Hybridoma. CloneMedia Hybridoma is an all-in-1 media which contains the semi solid concentrate + media that has been optimized to support hybridomas growth in the matrix. If the routinely used liquid media differs greatly in composition from CloneMedia Hybridoma, then a period of adaptation to the media may be required.
- Add the fluorescent detection reagent, CloneDetect, to the media at the same time. This will add minimal volume to the final concentration (1 mL in 100 mL).
- Incubate plates with solid cultures at 37 ºC and 5% CO2, leaving undisturbed for at least 4 days.
- Verify the presence of growing colonies at a suitable time point using a light microscope, usually ~10 days. Determine the approximate number of colonies/well. Ideally plates should obtain ~100 colonies/well.
- If growth was not obtained, it may be necessary to add supplementation. For a stable line, we recommend initially trying:
- FBS/FCS. Increase serum up to 20-30% final concentration. Our CloneMedia Hybridoma contains 20%.
Establish hybridomas screening using fresh fusion
Purpose: To optimize conditions in semi solid media using a fresh fusion. When plating fresh hybridoma fusions the optimal seeding density is highly dependent on the fusion efficiency as well as the kinetics and efficiency of selection. Cells may require a period of recovery prior to seeding in semi solid media. For the successful selection of hybrids, the selection pressure applied (i.e. concentration of HAT) may also require optimization.
- Let the fusion rested for 24-48 hrs in liquid selection “HAT media” prior to plating in semisolid media.
- Using the same media optimized for the established hybridomas, plate to 100 mL final volume. Supplement with 1X HAT, preferred antibiotic, and L-Glutamine final concentration 8mM. (Recommend Invitrogen’s GlutaMax, which is the more stable analogue.
- On the day of plating, spin to concentrate cells to 2 mL. Obtain a count of total viable cells.
- Plate cells at 4 different densities: 5000, 1X104, 1X105, 1X106 cells/mL.
- Incubate plates for at least 7 days at 37°C and 5% CO2, leaving undisturbed for at least 4 days.