Hello, Juliane!

Article LPS induced inflammatory responses in human peripheral blood...

Bacterial LPS has been extensively used in models studying inflammation as it mimics many inflammatory effects of cytokines, such as TNF-α, IL-1β or IL-6. The cellular receptor transducing the LPS signal has been identified as Toll-like receptor 4 (TLR4) [3–5]. Binding of LPS to TLR4 leads to the activation of NF-κB through the recruitment and activation of MyD88, IL-1R kinase (IRAK), TNFR associated factor 6 (TRAF-6), as well as NADPH oxidase (Nox) [2, 6, 7]. NF-κB plays a crucial role in regulating the transcription of genes related to innate immunity and inflammation responses and several studies indicate its activation is controlled by reactive oxygen species (ROS) in immune modulation in the lungs and in monocytes [8–11]. Several studies searching for novel anti-inflammatory agents have led to the identification of a key role for phosphatidylinositol 3-kinase (PI3K) in transducing receptor-mediated signalling during inflammation.

All these events activate PBMCs, promote their proliferation, in which cytoskeletal proteins and integrins involved in cell differentiation and adhesion are inhibited.

Hello Alexandr,

thank you for your answer! So does it mean, that because of activation monocytes get less adherent?

LPS stimulation of monocytes:

You will get a macrophage population of M1 types and it's inflammatory.

Monocytes are myeloid lineages and activates with LPS.

Please use non heat killed FCS if you are working with any bacterial infection model because it will not interfere with opsonization...Thanks.

I am agreed with Babak and it's important to see CD11b (adherence marker expression) and HLA-DR marker (in case you are maturing to macrophages).

Non heat killed Featal calf serum enhance adhearance..

It depends on microenvironment which LPS provides.

Usually when we perform experiments on monocytes, we allow monocytes to adhere on surface (we it any well plate) by leaving them in complete medium for overnight or incomplete medium for 5-6 hours. And then we change the media with desired supplements (LPS, MCSF, GMCSG, Any ILs).

If you are adding your supplements at the starting of PBMCs (not allowing monocytes to adhere) then due to present microenvironment if they will be differentiating in DC, they will not adhere or if the environment differentiating towards Macfophage, then they will adhere.

As per my experience.

First let the monocytes to be attached on the well surface overnight, then change the media with LPS. It might help in better adherence. All the best!

Hello Babak, sorry for my late answer. We indeed incubated the monocytes for one hour at 37°C to get them adherent and then added 1µl (50ng/ml). Before Addition of LPS, cells were adherent and after incubation with LPS even after 15 minutes, cells were in suspension again. before this experiment, we worked with LPS unstimulated cells and they stayed adherent.