2 replies to this topic

[radioactive]

Hi everyone,

I am doing clonogenic assay on primary cells but after two attempts, I only got 5% plating efficiency. One suggestion from an article that I found was to add conditioned media to the regular media. How does one prepare conditioned media? What I have done so far was took the media from growing culture of same cells and centrifuged it down to removed cell and cell debris. Then I am planning to add this to the regular media in a 1:1 ratio.  Nobody in the lab has done this before, any suggestion would be greatly appreciated.

[lab rat]

Hi Radioactive,

We have used conditioned medium for hybridoma cell lines. Your dilution of 1:1 may be too much.

We centrifuge down the cells, aspirate the supernatant, and store it at 4C with 0.2% NaAz. We add between 100-200 ul of conditioned medium per 10 ml of fresh medium. This small amount of azide doesn't seem to hurt our cells, but you may want to omit the azide for your primaries.

Regards,

lab rat

[radioactive]

lab rat, on Aug 24 2009, 02:01 PM, said:

Hi Radioactive,

We have used conditioned medium for hybridoma cell lines. Your dilution of 1:1 may be too much.

We centrifuge down the cells, aspirate the supernatant, and store it at 4C with 0.2% NaAz. We add between 100-200 ul of conditioned medium per 10 ml of fresh medium. This small amount of azide doesn't seem to hurt our cells, but you may want to omit the azide for your primaries.

Regards,

lab rat

Hi lab rat,

Thank you for the reply. I'll try this!

radioactive