Protocol for Cell Cloning by Serial Dilution in 96 Well Plates

This technique is widely used for the clonal isolation of hybridomas and other cell lines that are not attachment-dependent. However, it is also handy for cloning attachment-dependent cells when the cell plating efficiency is very low, unknown, or unpredictable. This method is fast and easy; however, like most clonal isolation methods, there is no guarantee that the colonies arose from single cells. Re-cloning a second time is advised to increase the likelihood that the cells originated from a single cell.

• Fill the reagent dispensing tray with 12 mL of the appropriate culture medium, then using an 8-channel micro pipette add 100 μL medium to all the wells in the 96 well plate except the well which is left empty.
• Add 200 μL of the cell suspension to the first well. Then using a single channel pipettor quickly transfer 100 μL from the first well to the second well and mix by gently pipetting. Avoid bubbles. Using the same tip, repeat these 1:2 dilutions down the entire column, discarding 100 μL from the last well so that it ends up with the same volume as the wells above it.
• With the 8-channel micro pipettor add 100 μL of medium to each well in column 1 (giving a final volume of cells and medium of 200 μL/well). Then using the same pipettor quickly transfer 100 μL from the wells in the first column to those in the second column and mix by gently pipetting. Avoid bubbles!
• Using the same tips, repeat these 1:2 dilutions across the entire plate, discarding 100 μL from each of the wells in the last column so that all the wells end up with 100 μL of cell suspension.
• Bring the final volume of all the wells to 200 μL by adding 100 μL medium to each well. Then label the plate with the date and cell type. Adding filtered conditioned medium (medium in which cells have been previously grown for 24 hours) to the wells can increase the success rate (cloning efficiency) for difficult-to-grow cells.
• Incubate plate undisturbed at 37°C in a humidified CO₂ incubator.
• Clones should be detectable by microscopy after 4 to 5 days and be ready to score after 7 to 10 days, depending on the growth rate of the cells. Check each well and mark all wells that contain just a single colony. These colonies can then be sub-cultured from the wells into larger vessels. Usually, each clone is transferred into a single well in a 12-well or 24-well plate.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides the world's most comprehensive list of cells and has realized that animal and human primary cells, tumor cell lines, continuous (immortalized) cell lines, and tissues are critical to the biopharmaceutical industry and biomedical research as reagents, therapeutic modalities, and as proxy materials.
• In recent years proof of clonality has become a focus. Many companies have received comments back on this topic during the IND review process of their biological products. We also provide the genetic characterization of producer cell lines by FISH, which offers information on transgene integrity and integration sites.

Transferring clones directly from a well in a 96-well plate into a T-25 flask is not recommended. The cells may be unable to grow due to their inability to condition the larger medium volume in the flask. Using some conditioned medium when subculturing the cells for the first time will also help them survive and grow.

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