Optimizing Plating Density in Semi-Solid Media for Hybridoma Selection

Hybridoma selection is a critical step in monoclonal antibody production. Prior to plating for selection, it is important to determine the optimal density, which can vary with cell lines and fusion conditions. If the culture dish is seeded at too high density, colony overcrowding may prevent the user from being able to pick discrete colonies. If the culture dish is seeded too low, the user ends up wasting media. In this technical tip, we outline how to determine an optimal plating density for hybridoma selection.

What is Hybridoma Selection?

Hybridoma selection, performed after the fusion of myelomas and spleen cells, is typically carried out in HAT (hypoxanthine, aminopterin, and thymidine)-containing media, which can be either liquid or semi-solid. Compared to liquid medium, a semi-solid selection medium allows for more discrete colony formation, as the cells remain localized. Each distinct colony can be harvested and screened for antibody production. The ClonaCell™-HY product line offers ClonaCell™-HY Liquid HAT Selection Medium, a liquid hybridoma selection medium containing HAT, and ClonaCell™-HY Medium D—a semi-solid medium for selecting and cloning hybridomas.

What Is the Optimal Plating Density for Hybridoma Selection?

The optimal concentration at which cells should be plated may vary depending on cell type, mouse strain, methodology, fusion efficiency, etc. We recommend plating at several cell densities when performing HAT selection in ClonaCell™-HY Medium D. The steps for determining an optimal plating density for hybridoma selection are described below.

  1. On the day after fusion, resuspend the fused cells in 5 mL of ClonaCell™-HY Medium C. This resuspension volume allows low, medium, and high plating conditions to be tested without exceeding the maximum liquid volume.
  2. To test 3 different cell densities, mix the cell suspension with ClonaCell™-HY Medium C & Medium D as indicated below:

    Cell Density
    Volume of fused cell suspension in ClonaCell™-HY Medium C (mL)
    Volume of ClonaCell™-HY Medium D (mL)
    Additional volume of ClonaCell™-HY Medium C required to top up to final volume (mL)
    Final volume (mL)
  3. Plate 9.5 mL per 100 mm culture dish (Non-Treated, Catalog #38045).
  4. It will take ~2 weeks to clearly see how many hybridoma colonies survive fusion and selection. Choose an optimal plating density depending on how crowded or sparse your surviving colonies are on the culture dish.


We recommend targeting 100 - 200 colonies per 100 mm culture dish. A culture dish with more than 200 colonies would be overcrowded, making it difficult to pick discrete colonies.

Products for Hybridoma Selection

Catalog #
ClonaCell™-HY Medium D
ClonaCell™-HY Medium D without HAT
ClonaCell™-HY Liquid HAT Selection Medium

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