which HDL function? HDL have anti-inflammatory, anti-oxidative, anti- aggregatory, anti-coagulant, and pro-fibrinolytic properties.

Anti-inflammatory

As I understand, you want to asses on some in vivo model the anti-inflammatory function of HDL, if this is true, I believe that this article could be helpful. But if what you want is something different, please try to explain a bit more your question.

thanks for your article Paola Ibelles

I just want to calculate HDL function in different serum samples (dyslipidemia patients) .At present am using DCFH (2,7-Dichlorodihydrofluorescein diacetate ) for this function test but i dont know how to calculate .

DCFH is used to measure the oxidative conditions on the assay rather than the inflammatory, as far as I know. I searched a little on DCFH and inflammation, but didn't find any information on it, and can't imagine what exactly you want to calculate. Is a coupled reaction? If it is, perhaps tha publication with the original methodology explains what and how to calculate

Alternatively, you may assess the antiinflammatory effect of HDL in cultured macrophages stimulated with lipopolysaccharide (LPS). To do so, you would first to isolate the HDL from total plasma from dyslipidemic and control subjects by ultracentrifugation.

The anti-inflammatory action of HDL is determined by adding them to monocyte-derived (THP-1) macrophages. After differentiation the macrophages are ready to be treated without or with the HDL (eg., 50 microg of protein per mL). Add the HDL in cell culture medium without FCS and following add the LPS (10 microg per mL). Incubate the cells with HDL in a final volume of 500 microL/well (24-multiwell plate) for 24 h. Following HDL incubation. After the incubation, recover the medium and the cells from each well and freeze the samples until interleukin analysis (eg. IL-6, MCP-1 or TNFalpha). Wash de cells with cold phosphate-buffered saline (PBS) twice and then scrape them in FCS-free medium or PBS.

Keep in mind to reserve some seeded wells to check the effect of basal effect of LPS compared with its absence. The effect of HDL should be assayed in the absence and the presence of LPS. Also check the suitable HDL concentration to carry out the experiments. HDL may be isolated with fresh tubes or if recycled especifically treated to avoid interference of bacterian lipotoxins.

Thank you Josep Julve .

Dear Josep Julve ,

Do you have any articles regarding ur protocol ? please send me if possible thank you.

Can i use HDL precipitation buffer instead of using ultra centrifugation ? for HDL separation.

HI Rex, in my opinion, preparative UC is the suitable method to obtain enough amount of HDL and to clearly assess a diorect HDL-related effect.

Hi again Rex, please find attached a review on HDL antiinflammatory assessment.

I have found another publication in which the effect of HDL has been determined using apoB-depleted serums. This other assay looks simple and may work.

Thank you so much Josep Julve .