Dear Jule,

there is one crucial issue when you stimulate with LPS: you have to be LPS-free to begin with.

This is often difficult to achieve because all your reagents and media have to be clean. Fetal calf serum added to the cultures often is contaminated and you need a batch that has been pretested. I good approach is to make your own human serum from blood donors.

Also, the adhesion step you use will already activate the cells and will interfere with the signalling in response to exogenous LPS.

In order to enrich for monocytes without adhesion you may want to try RosetteSep from StemCell Technologies, which is a no-touch procedure.

With all these problems we have worked with cell lines like Mono Mac 6 in man

and P338D1 in the mouse. Here you have pure monocytes to beginn with and this is advantageous when you want to do molecular studies.

Coming back to your observation: it may well be that your purification has already initiated the NF-kB signalling cascade and is beyond the peak and on the decline, additional LPS will speed up the decline.

Anyway, there is no simple solution to your problem.

In case you want to go for cell lines, then go for Mono Mac 6 (DSMZ cell bank). I have enclosed a couple papers for your information.

Best of luck

Loems

Thanks a lot Loems for your detailed answer.

I have one question concerning your recommendation: if i used Rosette sep for purification, how is this method able to prevent adherence on the plates? As I read, Rosette just offers negative and quick selection?

best regards!

Jule

Dear Jule,

the RosetteSep procedure only involves density gradient centrifugation, so there is no adherence step.

When you in the end want to treat the cells in wells or tubes then there is some adherence possible once the cells settle.

This does not generate much interference and the cells can be harvested for analysis by vigorous pipetting or vortexing.

Yours

Loems

thank you :)

Hi Jule,

I would add to this the question of whether you want to adhere your cells? If you do, they ought to be left in the plates to adhere at least overnight. Thy will begin to differentiate then though. We used to do this well before any additional stimulus, so that the cells have recovered from the adherence process and the activation which it induces.

We also used commercial FCS and medium, but as Loems says, it must be tested for endotoxin first. You can buy low-endotxin products which come with a certificate of endotoxin content. They must still be tested in practice and if the cells were not activated by the product we would then reserve that particular charge.

It's a long time since I personally did any of this, but I hope this helps a bit too :)

Best wishes,

Sharon