The easy way to prepare conditioned media is to centrifuge a particular volume of the culture (the amount you need) in the particular metabolic phase with high speed rpm, discard the pellet and filter sterilise the supernatant with 0.45 μm membrane filter, store the conditioned medium in a freezer until use.

Best wishes.

Satyendra Mondal gave you the protocol for generating the conditioned media, but as to the right growth conditions, media, length of growth, those will need to be experimentally determined. It is unlikely that any laboratory growth condition is going to recapitulate in vivo physiological conditions but that may not necessarily matter.

Satyendra Mondal and Michael J. Benedik thank you both for your answers.

I am also following a similar protocol for the generation of conditioned media and currently standardizing the time points in which I should collect the conditioned media.

As for the media I was wondering whether you would culture the bacteria in their own media (etc. LB) or in the media in which I will add the generated media to the cancer cells (etc. RPMI). I am mainly interested in the effect of the bacterial metabolites to the cancer cells.

Thanks again for your comments, much appreciated!

There is no real way to predict the effect of media without doing some trial and error, but my guess is that it won't matter. Without knowing how (or if) the factor you are looking for is regulated, then it is all just guesswork at this point.

Ioanna Nikdima I do not have expertise in this field but the attached research article used the bacterial media (LB) for the preparation of conditioned media. Please check the related papers in this field.

Article i>E. coli Secretome Metabolically Modulates MDA-MB-231 Breas...

thank you Satyendra Mondal !!

thank you Michael J. Benedik !!

هناك عدة اوساط زراعية تستخدم في معرفة الافرازات الايضية للبكتريا المراد تشخيص المواد المنتجة منها والتي نباع في مراكز خاصة للاحياء المجهرية

Salih Khalaf thank you for your reply I will have a look!