Dear Xiarong,

I had some success with using SeaKem low melting point agarose with normal culture media to give appropriate soft agar for isolating clonal populations of (normally) suspension haematopoietic cell lines. I made the agarose to an optimal concentration for each cell line (around 0.2%), let the cells form colonies for around 10-14 days before isolating. My method for this was rather unusual - i found that it was difficult to 'suck up' the colonies directly with a normal pipette and tip, as the colonies often scattered with only some cells going into the pipette tip. I therefore found if you put the tip the 'other way round' in a Gilson p200, leaving the end with the larger area free - you press it into the agarose, rotate slightly and use the pipette to provide a little bit of suction to essentially 'cut' out the disc of agar containing your colony. I then transferred these 'discs' into normal liquid media in 96 (or 48) well plates and allowed the clones to proliferate-gradually transferring the cells to larger media volumes to get the required number of cells. If you send me a message with your email address, i can send you the protocol (although it is rather short, and there is not much more detail than i include here). Kind regards and good luck.

Vicky

Dear Xiarong,

I had some success with using SeaKem low melting point agarose with normal culture media to give appropriate soft agar for isolating clonal populations of (normally) suspension haematopoietic cell lines. I made the agarose to an optimal concentration for each cell line (around 0.2%), let the cells form colonies for around 10-14 days before isolating. My method for this was rather unusual - i found that it was difficult to 'suck up' the colonies directly with a normal pipette and tip, as the colonies often scattered with only some cells going into the pipette tip. I therefore found if you put the tip the 'other way round' in a Gilson p200, leaving the end with the larger area free - you press it into the agarose, rotate slightly and use the pipette to provide a little bit of suction to essentially 'cut' out the disc of agar containing your colony. I then transferred these 'discs' into normal liquid media in 96 (or 48) well plates and allowed the clones to proliferate-gradually transferring the cells to larger media volumes to get the required number of cells. If you send me a message with your email address, i can send you the protocol (although it is rather short, and there is not much more detail than i include here). Kind regards and good luck.

Vicky

Dear Victoria Forster - As I read your post about soft agar assay, I have a question to ask. Do you also do cell survival assay like clonogenic assay , Would you happen to know any technique to determine clonogenicity of cells in suspension (JURKAT)?

Hi Minakshi, yes i did also use the soft agar technique for doing clonogenic assays. I did not use JURKAT, but had some success with other haematopoietic cell lines such as HL-60 using this method. I think as long as you optimise the technique to use the correct concentration of agarose for each cell line you use, to ensure maximum cloning efficiency, you could use it as an assay. However, i would suggest that this assay will not pick up very small changes in cloning efficiency - as even if you are exceptionally careful in weighing out the agarose and diluting it with the media etc, agarose concentrations vary between batches and even small changes in this can very markedly affect cloning efficiency. I hope this helps.

Dear Victoria Forster

As I read your post about soft agar assay, I have looked for how to recover cells from soft agar. Would you send your protocols to me?? If you don't mind please send me your protocol by e-mail. My e-mail address is [email protected].

Hi Victoria,

I am wondering if you are still able to share your protocol for recovering cells from Soft agar formation assay or not. I am in a project that require me to figure out a way to isolate genomic DNA from the cells in soft agar condition.

Please email to [email protected] .

Thanks ! -- Bo

Victoria J Forster Minakshi Gandhi ,

If you cut out the soft agar and place it in normal media, will the colony distribute in whole media. I want to analyse the protein expression and do Flow Cytometry in those colonies. May I know the downstream procedure.

Thanks for the above answers.