ClonaCell™-CHO ACF is a methylcellulose-based semi-solid medium with supplements for the selection and cloning of suspension-adapted CHO cells in serum-free conditions. This medium is animal component-free and contains only recombinant proteins and synthetic components. Single-cell cloning efficiency of CHO cells is higher in this medium than in protein-free media, especially at low plating densities. It does not contain L-glutamine, selection agents, or phenol red and is compatible with dihydrofolate reductase (DHFR) and glutamine synthase (GS) selection systems.
Benefits of semi-solid cloning:
• Individual cells are suspended in viscous medium and form physically separated, discrete colonies that are easily isolated.
• Monoclonal cell lines are isolated in less time using fewer resources compared with selection and cloning by limiting dilution.
• Diverse clones with a wide range of growth rates and productivities form discrete colonies in the viscous medium. As a result, rare and high-producing clones can be individually isolated more easily using simultaneous selection and cloning in semi-solid medium compared with selection in bulk liquid cultures.
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Why do I get more cells when I select my fusion in liquid medium rather than in methylcellulose-based semi-solid medium?
Cells grown in liquid medium may appear to grow more rapidly than in methylcellulose-based medium. This is often due to the presence of a few rapidly growing clones that multiply quickly and become abundant in liquid culture, overgrowing clones that grow more slowly. In methylcellulose cultures, the rapidly growing cells remain in close proximity to each other, resulting in large colonies, each derived from a single fusion or transfection product. The large clones don't overgrow smaller, slower growing colonies, which can be separately isolated.
How do I thaw ClonaCell™ methylcellulose-based semi-solid medium?
We recommend thawing the medium overnight in a refrigerator at 4°C and mixing well.
How do I measure and dispense methylcellulose semi-solid medium?
We recommend using a 12 mL syringe with a 16 gauge needle attached (blunt end needles are recommended for safety purposes). Do not dispense the semi-solid media/cell mixture using serological pipettes as the media will stick to the pipette walls, resulting in inaccurate dispensed volumes and loss of cells.
My ClonaCell™ methylcellulose semi-solid medium appears runny. Why does this happen?
"Runny" methylcellulose could be a result of improper handling. Diluting the methylcellulose with too much liquid medium, or insufficient mixing before use, will result in methylcellulose with altered viscosity. Excessive condensation on the inside of the cell culture dish lid can result in water dripping onto the cultures, lowering viscosity. Additionally, bumping, shaking or other sudden movement of the culture may also disrupt the colonies. Note: methylcellulose is less viscous at room temperature than at 37°C.
What is the optimal number of colonies per plate?
We recommend 50-150 colonies per plate. As it is difficult to anticipate the numbers of colonies after a fusion or transfection, we recommend plating at three different densities to increase the likelihood of achieving a plating density of approximately 100 colonies per plate. This density allows sufficient space between the colonies to allow for easy colony picking.
There are still bubbles in the media after I plate my cells. Do I need to disrupt the bubbles?
We recommend that you avoid creating large bubbles during plating, but there is no need to manually pop or disperse the small bubbles after plating. They will disperse over the incubation period of 10-14 days.
Do I ever need to re-clone cultures grown with ClonaCell™ semi-solid medium?
Re-cloning is a good practice to observe and is recommended if the number of colonies in the original dishes was very high.
Once I pick the colonies and grow the cells in plates, will the residual methylcellulose interfere with characterization? For example, will I have problems doing an ELISA?
There will likely be some residual methylcellulose contamination when colonies are picked and transferred to the 96-well plate with the liquid growth medium. The concentration of methylcellulose, however, should be low enough that it should not interfere with most assays.
How important is the incubator humidity when culturing in methylcellulose-based medium?
Very important. In situations where the humidity is not high enough, we recommend that the 100 mm Petri dishes should be placed with an open dish containing sterile water inside a larger plastic container with a lid. Without very high humidity, the media will dry out over the culture period and this will impede the growth of the colonies.
Do I have to use 100 mm petri dishes or can I use other cultureware?
We recommend 100 mm Petri dishes as these have been used to develop and test ClonaCell™ semi-solid media. We have found that the surface area of these dishes allows for easy colony picking. Other sizes of dish (e.g. 6-well plates) can be used. It is important to use non-coated dishes to prevent cells from sticking to the bottom of the plate and obscuring the colonies. The volume of media plated should be adjusted to reflect the surface area of the dish being used.
Are there any selective agents in ClonaCell™-CHO ACF or ClonaCell™-CHO CD?
These media do not contain hypoxanthine, aminopterin, thymidine, glutamine, or any other selective agents or antibiotics. These media are therefore fully customizable. Both ClonaCell™-CHO ACF and ClonaCell™-CHO CD are compatible with dihydrofolate reductase (DHFR) and glutamine synthetase (GS) selection.
My cells grow fine in ClonaCell™-CHO ACF but I do not see colony growth in ClonaCell™-CHO CD. Why?
The plating density for successful colony growth in protein-free medium needs to be higher than what is recommended for medium that contains protein. The optimal plating density should be determined empirically by plating several cell densities. For example, when re-cloning established cell lines, we recommend testing 200-1000 cells per 100 mm plate for ClonaCell™-CHO ACF and testing 2000-10000 cells per 100mm dish for ClonaCell™-CHO CD.
What medium do you recommend for the expansion of clones after selection in ClonaCell™-CHO ACF or ClonaCell™-CHO CD?
CHO cells selected in either ClonaCell™-CHO ACF or ClonaCell™-CHO CD semi-solid medium can be expanded in ClonaCell™-CHO CD Liquid medium.
How many cells should I plate for cloning in ClonaCell™-CHO CD?
Cloning efficiencies vary for different clones and cell lines. For optimal cloning efficiencies, cells should be in logarithmic phase prior to plating. If you have had previous experience with cloning in a chemically defined liquid medium, plating cell concentration that would result in ~100 clones may be used to plate in semi-solid medium (e.g. If plating at 1 cell/well in a 96-well plate results in 10 wells being positive for clones, plating 1000 cells per 100mm plate in semi-solid medium would be a good starting cell concentration to test). A minimum of three plating cell concentrations is recommended. If no work has been previously done with the specific cell line, we recommend a range of 25 000 - 100,000 cells per 100 mm dish for selection and cloning after transfection, and 2000 - 10,000 cells per 100 mm dish for subcloning. Additional optimization of cell concentrations may be necessary.
Is the serum in ClonaCell™-TCS medium heat inactivated?
Yes, all serum used in ClonaCell™ is heat inactivated.
Is there any IgG in ClonaCell™ TCS?
While we don't add IgG to the ClonaCell™ media, we do add serum, which contains an undefined amount of IgG. We selectively use serum lots with low IgG levels in the production of ClonaCell™ media, however, levels vary from lot to lot. IgG levels in a specific lot of ClonaCell™ TCS medium are available in the lot-specific Certificate of Analysis.
Can ClonaCell™-TCS be used with any cell line?
A list of recommended cell lines can be found in the manual. Other cell lines may be compatible with ClonaCell™-TCS. It will be necessary, however, to determine the plating cell density and growth efficiency of the desired cells in ClonaCell™-TCS.
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