Hi, how do you get your hybridoma cells to grow as neat colonies and not as a long elongated clump at the edge of the well?
Are you referring to the first few days after fusion? After fusion there are normally clumps with a lot of debris. After about 5-7 days a person can see colonies appearing in the debris.
Are you referring to immediately after fusion? Immediately after fusion there are normally a lot of clumps with cell debris. After 5-7 days colonies become clearly visible in the clumps & debris. By about 10 days the wells should be covered with hybridomas.
Thank you for your replies.
This happens during the process of subcloning. I strongly assume that these long elongated "colonies" grow from more than one cell only because they are far too big. So if the antibody-producing cells are buried within this long "colony" it makes picking them impossible. This is the reason I would like the hybridomas to grow in distinct colonies.
Hi I should not happen during sub-cloning. A person will normally see a single cell that divides at a rate of approx doubling time 15-24 hrs. After 5 days normally a very distinct colony with numerous cells with a very good morphology. Could this be due to swirling of the dish at medium addition? Rotational force will take cells to the outside and swirling motion will then disperse them in elongated pattern.
Thank you, Edmund Pool, for your answer.
The 96-well plates which are used for subcloning are not swirled. But I think you are right. It must be some motion of the medium that causes it. Addition of medium is done with a multi-channel pipette at an 45° angle. A different angle or short shaking of the plate might make a difference.
Our hybridoma grew as elongated lumps on the edge of wells because the drawers of the incubator were not even. We have corrected that now. Thank you again for all your suggestions.
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